Normoxic Animals Research Articles

Functional, structural, and molecular remodelling of the goldfish (Carassius auratus) heart under moderate hypoxia.

The goldfish (Carassius auratus) is known for its physiologic ability to survive even long periods of oxygen limitation (hypoxia), adapting the cardiac performance to the requirements of peripheral tissue perfusion. We here investigated the effects of short-term moderate hypoxia on the heart, focusing on ventricular adaptation, in terms of hemodynamics and structural traits. Functional evaluations revealed that animals exposed to 4days of environmental hypoxia increased the hemodynamic performance evaluated on ex vivo cardiac preparations. This was associated with a thicker and more vascularized ventricular compact layer and a reduced luminal lacunary space. Compared to normoxic animals, ventricular cardiomyocytes of goldfish exposed to hypoxia showed an extended mitochondrial compartment and a modulation of proteins involved in mitochondria dynamics. The enhanced expression of the pro-fission markers DRP1 and OMA1, and the modulation of the short and long forms of OPA1, suggested a hypoxia-related mitochondria fission. Our data propose that under hypoxia, the goldfish heart undergoes a structural remodelling associated with a potentiated cardiac activity. The energy demand for the highly performant myocardium is supported by an increased number of mitochondria, likely occurring through fission events.

Nicotinamide Riboside, an NAD + Precursor, Protects Against Cardiac Mitochondrial Dysfunction in Fetal Guinea Pigs Exposed to Gestational Hypoxia.

Gestational hypoxia inhibits mitochondrial function in the fetal heart and placenta contributing to fetal growth restriction and organ dysfunction. NAD + deficiency may contribute to a metabolic deficit by inhibiting oxidative phosphorylation and ATP synthesis. We tested the effects of nicotinamide riboside (NR), an NAD + precursor, as a treatment for reversing known mitochondrial dysfunction in hypoxic fetal hearts. Pregnant guinea pigs were housed in room air (normoxia) or placed in a hypoxic chamber (10.5%O2) for the last 14days of gestation (term = 65days) and administered either water or NR (1.6mg/ml) in the drinking bottle. Fetuses were excised at term, and NAD + levels of maternal liver, placenta, and fetal heart ventricles were measured. Indices of mitochondrial function (complex IV activity, sirtuin 3 activity, protein acetylation) and ATP synthesis were measured in fetal heart ventricles of NR-treated/untreated normoxic and hypoxic animals. Hypoxia reduced fetal body weight in both sexes (p = 0.01), which was prevented by NR. Hypoxia had no effect on maternal liver NAD + levels but decreased (p = 0.04) placenta NAD + levels, the latter normalized with NR treatment. Hypoxia had no effect on fetal heart NAD + but decreased (p < 0.05) mitochondrial complex IV and sirtuin 3 activities, ATP content, and increased mitochondrial acetylation, which were all normalized with maternal NR. Hypoxia increased (p < 0.05) mitochondrial acetylation in female fetal hearts but had no effect on other mitochondrial indices. We conclude that maternal NR is an effective treatment for normalizing mitochondrial dysfunction and ATP synthesis in the hypoxic fetal heart.

Prematurity and Hyperoxia Have Different Effects on Alveolar and Microvascular Lung Development in the Rabbit.

Bronchopulmonary dysplasia (BPD) is a developmental disorder of infants born prematurely, characterized by disrupted alveolarization and microvascular maturation. However, the sequence of alveolar and vascular alterations is currently not fully understood. Therefore, we used a rabbit model to evaluate alveolar and vascular development under preterm birth and hyperoxia, respectively. Pups were born by cesarean section 3 days before term and exposed for 7 days to hyperoxia (95% O2) or normoxia (21% O2). In addition, term-born rabbits were exposed to normoxia for 4 days. Rabbit lungs were fixed by vascular perfusion and prepared for stereological analysis. Normoxic preterm rabbits had a significantly lower number of alveoli than term rabbits. The number of septal capillaries was lower in preterm rabbits but less pronounced than the alveolar reduction. In hyperoxic preterm rabbits, the number of alveoli was similar to that in normoxic preterm animals; however, hyperoxia had a severe additional negative effect on the capillary number. In conclusion, preterm birth had a strong effect on alveolar development, and hyperoxia had a more pronounced effect on capillary development. The data provide a complex picture of the vascular hypothesis of BPD which rather seems to reflect the ambient oxygen concentration than the effect of premature birth.

Upregulation of endothelial hypoxia‐inducible factor‐2 alpha in animals with pulmonary hypertension

IntroductionIdiopathic pulmonary arterial hypertension (IPAH) is a life‐threatening disease with no cure. Pulmonary vascular remodeling due to increased pulmonary artery smooth muscle (PASMC) and lung vascular endothelial cell (LVEC) proliferation is a major cause for the elevated pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) in patients with IPAH. We have previously demonstrated that endothelial hypoxia‐inducible factor‐2α (HIF‐2α) contributes to the development of pulmonary vascular remodeling in LVEC isolated from IPAH patients. Moreover, HIF‐2α is involved in the initiation of endothelial‐to‐mesenchymal transition (EndMT) by upregulating Snai1 and Snai2 (EndMT drivers). However, the exact role of HIF‐2α in the development and progression of pulmonary hypertension (PH) remains elusive. We aimed to investigate whether endothelial HIF‐2α is involved in pulmonary vascular remodeling in animals with hypoxia/sugen‐induced PH (SuHx‐PH) and monocrotaline‐induced PH (MCT‐PH).MethodsHypoxia/sugen‐PH (SuHx‐PH) model in mice was induced by exposing animals to the combination of normobaric hypoxia (10% O2) and sugen 5416 (20 mg/kg, i.p., weekly) for 6 weeks. Monocrotaline‐MCT‐PH model in rats was induced by single administration of MCT (60 mg/kg, s.c.) followed by experimental measurements after 28 days. Body weight (BW) was collected from all animals weekly. Hemodynamic parameters including right ventricular systolic pressure (RVSP), mean pulmonary arterial pressure (mPAP), the Fulton index (FI), right ventricular (RV) contractility, heart rate (HR) were measured to establish PH. The pulmonary artery (PA) wall thickness was estimated by H&amp;E staining. The protein expression of Snai1 and HIF‐2α was determined by Western blot and immunocytochemistry. Normal human LVEC was treated with TGF‐β1 for 7 days to induce EndMT. LVEC isolated with patients with IPAH was treated with HIF‐2α inhibitor Compound 2 for 8‐48 hrs.ResultsExposure of mice to the SuHx (6 weeks) or rats to MCT (4 weeks) resulted in the significant increase of RVSP, mPAP, RV contractility, and FI compared to control animals. We did not detect any changes in HR between corresponded groups. BW was decreased by 7% after the first week of the SuHx treatment in mice. By the end of 6 weeks BW was significantly lower by 12% in SuHx mice compared with normoxic animals. BW in MCT‐treated rats was 5% lower after the first week and at the end of experiment compared to vehicle‐treated animals. Pulmonary vascular remodeling estimated by PA wall thickness was enhanced in both SuHx‐PH mice and MCT‐PH rats. The protein expression of HIF‐2α and Snai1 was higher in lung tissues isolated from SuHx‐PH mice and MCT‐PH rats compared to controls. Moreover, HIF‐2α and Snai1 was activated in normal human LVEC treated with TGF‐β1 (10ng/ml) for 7 days. Additionally, HIF‐2α inhibitor, Compound 2 (30µM) was able to downregulate Snai1 protein expression in LVEC isolated from IPAH patients after 16 hours.ConclusionsOur results indicate that HIF‐2α is involved in pulmonary vascular remodeling in animals with PH. Our studies support the premise that targeting HIF‐2α would be a novel therapeutic approach for the treatment of IPAH and other forms of PH.

Diminished Pulmonary Arterial Smooth Muscle Cell Membrane Cholesterol Unmasks EGFR‐dependent Vasoconstriction

IntroductionChronic hypoxia (CH) leads to increased pulmonary vascular resistance and resultant pulmonary hypertension (PH) in patients with chronic obstructive pulmonary disease, sleep apnea, or in residents at high altitude. Recent studies from our laboratory have demonstrated that CH augments vasoconstrictor reactivity to a variety of stimuli, including pulmonary arterial smooth muscle cell (PASMC) stretch, membrane depolarization, and stimulation of G protein‐coupled receptors. This effect of CH is mediated by a switch in PASMC signaling from Ca2+‐dependent mechanisms towards a novel epidermal growth factor receptor (EGFR)‐NADPH oxidase 2 (NOX2)‐Rho kinase (ROK) Ca2+ sensitization pathway. The overall objective of this study is to understand the mechanism by which CH mediates this switch in signaling. We have recently demonstrated that CH decreases membrane cholesterol, a component of lipid membrane domains that can regulate both EGFR and NOX2 activity in other cell types. Therefore, we hypothesized that this EGFR‐NOX2‐ROK signaling pathway is intact in PASMC from control animals but quiescent due to inhibition by membrane cholesterol.MethodsWe first validated our ability to manipulate membrane cholesterol levels by treating primary cultures of PASMCs from Sprague Dawley rats with either cholesterol (2 mM), epicholesterol (an epimeric form of cholesterol; 2 mM), or MβCD (cholesterol chelator; 10 mM). Membrane cholesterol content was quantified by filipin (50 mg/ml) fluorescence using confocal microscopy. Subsequently, we examined vasoconstrictor responses to increasing concentrations of epidermal growth factor (EGF) (10‐10 – 10‐6 M) in pressurized, endothelium‐disrupted rat pulmonary arteries (~150 mm diameter). The effect of diminished membrane cholesterol on vasomotor responses was tested by substituting native cholesterol with epicholesterol (2 mM), which lacks the ability participate in protein‐sterol interactions while maintaining normal membrane fluidity. Vessels were additionally treated with the selective NOX2 inhibitor gp91ds‐tat (50 mM), the selective Rho kinase inhibitor fasudil (10 mM), or their respective vehicles.ResultsCholesterol supplementation significantly increased PASMC membrane cholesterol as indicated by filipin fluorescence. Conversely, treatment with either MβCD or epicholesterol significantly decreased membrane cholesterol compared to vehicle‐treated cells (Figure 1). In agreement with our hypothesis, epicholesterol‐dependent substitution of cholesterol revealed concentration‐dependent vasoconstriction to EGF in pulmonary arteries (Figure 2A). Treatment with either fasudil (Figure 2A) or gp91ds‐tat (Figure 2B) attenuated this effect.ConclusionsThe EGFR‐NOX2‐RhoA signaling cascade is present in arteries from normoxic animals but is normally inhibited by membrane cholesterol. The results of this study will allow for improved mechanistic analysis of how cholesterol regulates this pathway that is central to enhanced vasoconstrictor reactivity in PH.

EC Membrane Cholesterol Content Regulates the Contribution of TRPV4 Channels in ACh‐induced vasodilation in Rat Gracilis Arteries

Numerous studies show that ion channel activity is sensitive to the level of membrane cholesterol, with cholesterol most often suppressing channel activity. Our previous work demonstrated that endothelial cell (EC) membrane cholesterol is reduced following 48‐hr of exposure to chronic hypoxia (CH). Moreover, CH exposure also couples endothelial Transient Receptor Potential Subfamily V Member 4 (TRPV4) channels to muscarinic receptor signaling through an endothelium‐dependent hyperpolarization (EDH)‐pathway not present in control animals. TRPV4 appears to provide activator Ca2+ for endothelial large‐conductance Ca2+‐activation K+ channels in this setting. Taken together, this work suggests that changes in endothelial cell membrane cholesterol mediate this CH‐induced increase in ion channel functionality. Interestingly, both BK and TRPV4 channels contain Cholesterol Recognition/Interaction Amino Acid Consensus (CRAC) Motifs that bind cholesterol through protein‐sterol interactions. It is essential to separate the effects of CH from the influence of EC membrane cholesterol changes related to hypoxic exposure. Thus, we hypothesize that acute manipulation of endothelial cell membrane cholesterol inversely determines the contribution of TRPV4 channels to endothelial‐dependent vasodilation. To test this hypothesis, vasodilation responses to acetylcholine (ACh) were determined in gracilis arteries from male Sprague‐Dawley rats using standard pressure myography. Rats were exposed to either ambient atmospheric (atm.) pressure or 48‐hrs of hypoxia (0.5 atm). Arteries were pretreated with N‐nitro‐L‐arginine and indomethacin. EC membrane cholesterol was depleted using MβCD and supplemented with MβCD‐cholesterol. In arteries from normoxic animals (a replete cholesterol state), inhibition of TRPV4 did not affect ACh‐induced vasodilation. However, TRPV4 channels contribute to ACh‐induced vasodilation in these arteries when EC membrane cholesterol is depleted with MβCD. In contrast, inhibition of TRPV4 channels attenuated ACh‐induced vasodilation in arteries from CH animals that exhibit lower EC membrane cholesterol than normoxic controls. EC cholesterol repletion with MβCD‐cholesterol in arteries from CH animals abolished the contribution of TRPV4 channels to ACh‐induced vasodilation. In conclusion, our results demonstrate that EC membrane cholesterol impedes the contribution of TRPV4 channels in EDH‐mediated dilation. These results provide additional evidence for the importance of plasma membrane cholesterol content in regulating intracellular signaling and vascular function.

The Analysis of Phagocytic Myeloid Cells in Low and High Fiber Fed Mice after Three Weeks of Hypoxia

Pulmonary hypertension (PH) is a devastating disease that is characterized by increased blood pressure (BP) in the pulmonary vasculature. Immune cells participate in adverse cardiovascular remodelling of pulmonary vessels and could represent targets for PH treatment. It has been shown that patients with PH have increased numbers of interstitial macrophages and dendritic cells in the lungs that originate from circulating immune cells. These myeloid immune cells migrate to the vessel wall and promote vascular hypertrophy. Attenuation of immune cell invasion into the vessel wall could potentially slow the progression of PH. One of the possible ways to reduce inflammation in the cardiovascular system is dietary modification with increased content of soluble fiber. Soluble fiber increases the level of short chain fatty acids (SCFAs) in the plasma which demonstrate anti‐inflammatory properties in systemic hypertension and cardiac overload. In different models of cardiovascular diseases high fiber (HFi) diet and SCFAs improve cardiovascular functions reducing the level of immune cells at the site of injury. The effects of HFi diet on the pulmonary circulation are unknown. This study establishes the effects of low (LFi) and HFi diet on macrophage and dendritic cell composition in the lungs in the model of hypoxia‐induced PH.C57bl/6j mice were fed with LFi and HFi diet five weeks before and three weeks during hypoxic exposure (10% O2). After that hematocrit, cecum, spleen and body weight were measured. Flow cytometry was used to identify the proportion of alveolar and interstitial macrophages and dendritic cells in the lungs.Hypoxic animals had increased hematocrit level compared to normoxic controls. Hematocrit did not differ between LFi fed animals and HFi fed animals in hypoxic conditions but in normoxia hematocrit was higher in LFi diet group. Cecum weight was significantly higher in HFi fed mice than in LFi fed mice whereas total body weight and spleen weight was not affected by diet composition. The percentage of alveolar macrophages (CD11b+CD11c+CD64+) did not differ between groups. Hypoxia increased the proportion of dendritic cells (CD11b+MHCll+CD24+) and reduced the proportion of interstitial macrophages (CD11b+MHCll+CD64+) among CD11b+MHC ll+ cells. Dietary fiber did not affect the proportion of dendritic cells and interstitial macrophages in normoxic and hypoxic animals.This data demonstrates that three weeks of hypoxia changes the proportion of CD11b+ dendritic cells and interstitial macrophages in the mouse lungs however soluble dietary fiber did not affect it.

Hypoxia Inhibits Subretinal Inflammation Resolution Thrombospondin-1 Dependently.

Hypoxia is potentially one of the essential triggers in the pathogenesis of wet age-related macular degeneration (wetAMD), characterized by choroidal neovascularization (CNV) which is driven by the accumulation of subretinal mononuclear phagocytes (MP) that include monocyte-derived cells. Here we show that systemic hypoxia (10% O2) increased subretinal MP infiltration and inhibited inflammation resolution after laser-induced subretinal injury in vivo. Accordingly, hypoxic (2% O2) human monocytes (Mo) resisted elimination by RPE cells in co-culture. In Mos from hypoxic mice, Thrombospondin 1 mRNA (Thbs1) was most downregulated compared to normoxic animals and hypoxia repressed Thbs-1 expression in human monocytes in vitro. Hypoxic ambient air inhibited MP clearance during the resolution phase of laser-injury in wildtype animals, but had no effect on the exaggerated subretinal MP infiltration observed in normoxic Thbs1−/−-mice. Recombinant Thrombospondin 1 protein (TSP-1) completely reversed the pathogenic effect of hypoxia in Thbs1−/−-mice, and accelerated inflammation resolution and inhibited CNV in wildtype mice. Together, our results demonstrate that systemic hypoxia disturbs TSP-1-dependent subretinal immune suppression and promotes pathogenic subretinal inflammation and can be therapeutically countered by local recombinant TSP-1.

Developing chicken cardiac muscle mitochondria are resistant to variations in incubation oxygen levels.

BackgroundChronic exposure to hypoxia during vertebrate development can produce abnormal cardiovascular morphology and function. The aim of this study was to examine cardiac mitochondria function in an avian model, the chicken, in response to embryonic development under hypoxic (15% O2), normoxic (21% O2), or hyperoxic (40% O2) incubation conditions.MethodsChicken embryos were incubated in hypoxia, normoxia, or hyperoxia beginning on day 5 of incubation through hatching. Cardiac mitochondria oxygen flux and reactive oxygen species production were measured in permeabilized cardiac fibers from externally pipped and 1-day post hatchlings.ResultsAltering oxygen during development had a large effect on body and heart masses of externally pipped embryos and 1-day old hatchlings. Hypoxic animals had smaller body masses and absolute heart masses, but proportionally similar sized hearts compared to normoxic animals during external pipping. Hyperoxic animals were larger with larger hearts than normoxic animals during external pipping. Mitochondrial oxygen flux in permeabilized cardiac muscle fibers revealed limited effects of developing under altered oxygen conditions, with only oxygen flux through cytochrome oxidase being lower in hypoxic hearts compared with hyperoxic hearts. Oxygen flux in leak and oxidative phosphorylation states were not affected by developmental oxygen levels. Mitochondrial reactive oxygen species production under leak and oxidative phosphorylation states studied did not differ between any developmental oxygen treatment.ConclusionsThese results suggest that cardiac mitochondria function of the developing chicken is not altered by developing in ovo under different oxygen levels.

The air-breathing Alaska blackfish (Dallia pectoralis) remodels ventricular Ca2+ cycling with chronic hypoxic submergence to maintain ventricular contractility.

The Alaska blackfish (Dallia pectoralis) is a facultative air-breather endemic to northern latitudes where it remains active in winter under ice cover in cold hypoxic waters. To understand the changes in cellular Ca2+ cycling that allow the heart to function in cold hypoxic water, we acclimated Alaska blackfish to cold (5 °C) normoxia or cold hypoxia (2.1–4.2 kPa; no air access) for 5–8 weeks. We then assessed the impact of the acclimation conditions on intracellular Ca2+ transients (Δ[Ca2+]i) of isolated ventricular myocytes and contractile performance of isometrically-contracting ventricular strips. Measurements were obtained at various contractile frequencies (0.2–0.6 Hz) in normoxia, during acute exposure to hypoxia, and reoxygenation at 5 °C. The results show that hypoxia-acclimated Alaska blackfish compensate against the depressive effects of hypoxia on excitation-contraction coupling by remodelling cellular Δ[Ca2+]i to maintain ventricular contractility. When measured at 0.2 Hz in normoxia, hypoxia-acclimated ventricular myocytes had a 3.8-fold larger Δ[Ca2+]i peak amplitude with a 4.1-fold faster rate of rise, compared to normoxia-acclimated ventricular myocytes. At the tissue level, maximal developed force was 2.1-fold greater in preparations from hypoxia-acclimated animals. However, maximal attainable contraction frequencies in hypoxia were lower in hypoxia-acclimated myocytes and strips than preparations from normoxic animals. Moreover, the inability of hypoxia-acclimated ventricular myocytes and strips to contract at high frequency persisted upon reoxygenation. Overall, the findings indicate that hypoxia alters aspects of Alaska blackfish cardiac myocyte Ca2+ cycling, and that there may be consequences for heart rate elevation during hypoxia, which may impact cardiac output in vivo.

Mitochondrial capacity and reactive oxygen species production during hypoxia and reoxygenation in the ocean quahog, Arctica islandica.

Oxygen fluctuations are common in marine waters, and hypoxia-reoxygenation (H-R) stress can negatively affect mitochondrial metabolism. The long-lived ocean quahog, Arctica islandica, is known for its hypoxia tolerance associated with metabolic rate depression, yet the mechanisms that sustain mitochondrial function during oxygen fluctuations are not well understood. We used top-down metabolic control analysis (MCA) to determine aerobic capacity and control over oxygen flux in the mitochondria of quahogs exposed to short-term hypoxia (24 h <0.01% O2) and subsequent reoxygenation (1.5 h 21% O2) compared with normoxic control animals (21% O2). We demonstrated that flux capacity of the substrate oxidation and proton leak subsystems were not affected by hypoxia, while the capacity of the phosphorylation subsystem was enhanced during hypoxia associated with a depolarization of the mitochondrial membrane. Reoxygenation decreased the oxygen flux capacity of all three mitochondrial subsystems. Control over oxidative phosphorylation (OXPHOS) respiration was mostly exerted by substrate oxidation regardless of H-R stress, whereas control by the proton leak subsystem of LEAK respiration increased during hypoxia and returned to normoxic levels during reoxygenation. During hypoxia, reactive oxygen species (ROS) efflux was elevated in the LEAK state, whereas it was suppressed in the OXPHOS state. Mitochondrial ROS efflux returned to normoxic control levels during reoxygenation. Thus, mitochondria of A. islandica appear robust to hypoxia by maintaining stable substrate oxidation and upregulating phosphorylation capacity, but remain sensitive to reoxygenation. This mitochondrial phenotype might reflect adaptation of A. islandica to environments with unpredictable oxygen fluctuations and its behavioural preference for low oxygen levels.

Brief Report: Case Comparison of Therapy With the Histone Deacetylase Inhibitor Vorinostat in a Neonatal Calf Model of Pulmonary Hypertension.

Pulmonary hypertension (PH) is an incurable condition in humans; driven by pulmonary vascular remodeling partially mediated by epigenetic mechanisms; and leading to right ventricular hypertrophy, failure, and death. We hypothesized that targeting chromatin-modifying histone deacetylases may provide benefit. In this Brief Report we describe case comparison studies using the histone deacetylase inhibitor vorinostat (suberanilohydroxamic acid, 5 mg/kg/day for the first 5 study days) in an established model of severe neonatal bovine PH induced by 14 days of environmental hypoxia. Echocardiographic, hemodynamic, and pharmacokinetic data were obtained in hypoxia-exposed (one each, vorinostat-treated vs. untreated) and normoxic vorinostat-treated control animals (n = 2). Echocardiography detected PH changes by day 4 and severe PH over 14 days of continued hypoxic exposure. RV dysfunction at day 4 was less severe in vorinostat-treated compared to untreated hypoxic calves. Cardioprotective effects were partially maintained following cessation of treatment through the duration of hypoxic exposure, accompanied by hemodynamic evidence suggestive of reduced pulmonary vascular stiffening, and modulated expression of HDAC1 protein and genes involved in RV and pulmonary vascular remodeling and pathological RV hypertrophy. Control calves did not develop PH, nor show adverse cardiac or clinical effects. These results provide novel translation of epigenetic-directed therapy to a large animal severe PH model that recapitulates important features of human disease.

Membranous Expression of Integrin α5β1 is Elevated in Endothelial Cells of Pulmonary Arterial Hypertension

Pulmonary Arterial Hypertension (PAH) is an incurable pathology characterized by remodeling of small pulmonary arteries leading to increased pulmonary vascular resistance and right ventricular heart failure. The endothelial dysfunction and endothelial-to-mesenchymal transition (EndoMT) observed in PAH contribute to the formation of plexiform lesions. Fibronectin is a commonly used molecular marker of EndoMT and is abundantly present in the plexiform lesions observed in human PAH. We have previously observed that pulmonary artery endothelial cells (PAECs) from the lesion forming Sugen/hypoxia rat model of PAH demonstrate increased proliferation and angiogenesis compared to PAECs from normoxic control animals. As the fibronectin receptor integrin α5β1 promotes endothelial proliferation and angiogenesis, we aimed to determine whether the expression of integrin α5β1 was elevated in PAH PAECs. Sugen/hypoxia rat PAECs (PAH PAECs) or normoxic control cells were received from the University of South Alabama. Initially, we observed an increase in mRNA expression of integrins α5, αV and β1 in PAH PAECs compared to control cells using a PCR screening array for molecular markers of epithelial-to-mesenchymal transition. Increased protein expression of the integrin α5, αV and β1 monomers were also observed in membrane fractions of PAH PAECs via western blot, compared to the cell membranes of control PAECs following a cell fractionation assay. To confirm the presence of the integrin α5β1 heterodimer at the membranes of PAH PAECs, co-immunoprecipitation of integrins α5 and β1 was performed from the membrane fractions of PAH and control PAECs. Following immunoprecipitation, western blot analysis demonstrated a strong coprecipitation of integrin α5 and β1 in membrane fractions of PAH PAECs, compared to weak coprecipitation in control PAECs. To confirm the increased membranous expression of integrin α5β1 in human tissue, human pulmonary endothelial cells from patients with idiopathic PAH (IPAH), familial PAH (FPAH) and control cells from failed donors (FD) were analyzed with an integrin-mediated cell adhesion array for cell surface expression of alpha and beta integrins. Human cells were provided by PHBI under the Pulmonary Hypertension Breakthrough Initiative (PHBI)*. Monomeric α5- and β1-mediated adhesion and heterodimeric α5β1-mediated adhesion were increased in IPAH and FPAH cells compared to FD control pulmonary endothelial cells. Collectively, these data strongly indicate that protein levels of the integrin α5β1 fibronectin receptor are increased at the cell membranes of pulmonary endothelial cells in PAH.

Inhaled vitamin A is more effective than intramuscular dosing in mitigating hyperoxia-induced lung injury in a neonatal rat model of bronchopulmonary dysplasia.

Prevention of bronchopulmonary dysplasia (BPD) in premature-birth babies continues to be an unmet medical need. Intramuscular vitamin A is currently employed in preterm neonates to prevent BPD but requires intramuscular injections in fragile neonates. We hypothesized that noninvasive inhaled delivery of vitamin A, targeted to lung, would be a more effective and tolerable strategy. We employed our well-established hyperoxia-injury neonatal rat model, exposing newborn rats to 7 days of constant extreme (95% O2) hyperoxia, comparing vitamin A dosed every 48 h via either aerosol inhalation or intramuscular injection with normoxic untreated healthy animals and vehicle-inhalation hyperoxia groups as positive and negative controls, respectively. Separately, similar vitamin A dosing of normoxia-dwelling animals was performed. Analyses after day 7 included characterization of alveolar histomorphology and protein biomarkers of alveolar maturation [surfactant protein C (SP-C), peroxisome proliferator-activated receptor (PPAR) γ, cholinephosphate cytidylyl transferase, vascular endothelial growth factor and its receptor, FLK-1, and retinoid X receptors (RXR-α, -β, and -γ], apoptosis (Bcl2 and Bax) key injury repair pathway data including protein markers (ALK-5 and β-catenin) and neutrophil infiltration, and serum vitamin A levels. Compared with intramuscular dosing, inhaled vitamin A significantly enhanced biomarkers of alveolar maturation, mitigated hyperoxia-induced lung damage, and enhanced surfactant protein levels, suggesting that it may be more efficacious in preventing BPD in extremely premature infants than the traditionally used IM dosing regimen. We speculate lung-targeted inhaled vitamin A may also be an effective therapy against other lung damaging conditions leading to BPD or, more generally, to acute lung injury.

Gestational Hypoxia Inhibits Pregnancy-Induced Upregulation of Ca2+ Sparks and Spontaneous Transient Outward Currents in Uterine Arteries Via Heightened Endoplasmic Reticulum/Oxidative Stress.

Hypoxia during pregnancy profoundly affects uterine vascular adaptation and increases the risk of pregnancy complications, including preeclampsia and fetal intrauterine growth restriction. We recently demonstrated that increases in Ca2+ sparks and spontaneous transient outward currents (STOCs) played an essential role in pregnancy-induced uterine vascular adaptation. In the present study, we hypothesize that gestational hypoxia suppresses Ca2+ sparks/STOCs coupling leading to increased uterine vascular tone via enhanced endoplasmic reticulum (ER)/oxidative stress. Uterine arteries were obtained from nonpregnant and near-term pregnant sheep residing in low altitude or acclimatizing to high-altitude (3801 m) hypoxia for ≈110 days. High-altitude hypoxia suppressed pregnancy-induced upregulation of RyR1 and RyR2 (ryanodine receptor 1 and 2) protein abundance, Ca2+ sparks, and STOCs in uterine arteries. Inhibition of Ca2+ sparks/STOCs with the RyR inhibitor ryanodine significantly increased pressure-dependent myogenic tone in uterine arteries from low-altitude normoxic pregnant animals but not those from high-altitude hypoxic pregnant animals. Gestational hypoxia significantly increased ER/oxidative stress in uterine arteries. Of importance, the hypoxia-mediated suppression of Ca2+ sparks/STOCs and increase in myogenic tone in uterine arteries of pregnant animals were reversed by inhibiting ER/oxidative stress. Of great interest, the impaired sex hormonal regulation of STOCs in high-altitude animals was annulled by scavenging reactive oxygen species but not by inhibiting ER stress. Together, the findings reveal the differential mechanisms of ER and oxidative stresses in suppressing Ca2+ sparks/STOCs and increasing myogenic tone of uterine arteries in hypoxia during gestation, providing new insights into the understanding of pregnancy complications associated with hypoxia.

Tracking the ancestral functions of erythropoietin: neuroprotection &amp; mitochondria

It has long been thought that erythropoietin (Epo) is exclusively involved in erythropoiesis, allowing erythroid progenitor cells to survive and mature through their antiapoptotic action. We now know that Epo in mammals has also other functions in the brain, playing key roles in the development, maintenance, protection, and repair of the nervous system. However, the recombinant human Epo (rhEpo) has neuroprotective effects in orthoptera insects (such as grasshoppers), and this effect appears to be mediated by the cytokine receptor‐like factor 3 (CRLF3), raising interesting questions about the evolutionary origin of the Epo signaling pathway and its role in invertebrate species. Taking into account that: 1) Epo in mammals modulates the mitochondrial oxidative phosphorylation and the production of reactive oxygen species (ROS) in several tissues, including the brain; and 2) that insects appeared during a geological period (Cambrian explosion) in which the atmospheric O2 was increasing and required the implementation of antioxidant systems at the cellular level; here we tested the hypothesis that activation of the “Epo‐like” system in the brain domestic crickets (Acheta domesticus) exposed to 6 % of hypoxia during 5 days, modulates mitochondrial functions for preventing against oxidative damages. To do so, we used our oxygraph‐2K system (OROBOROS) that measures the mitochondrial bioenergetics in saponin‐permeabilized tissue of 2 mg weight. Our preliminary results showed that rhEpo increased the survival of domestic crickets exposed to hypoxia by 20%. We showed also that, in normoxic animals, rhEpo increased the mitochondrial O2 consumption rate (OCR), but in hypoxic animals, rhEPO limited the increase of mitochondrial OCR. In parallel, rhEpo significantly decreased the production of ROS in hypoxia. These preliminary results suggest that rhEpo significantly improves cricket’s survival under hypoxia, by promoting a robust antioxidant effect through mitochondria. Our data also suggest that a neuroprotective “Epo‐like” endogenous molecule evolved during the “Cambrian explosion” from a urbilaterian (common to vertebrates and invertebrates) ancestor.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada

Proteomic approach to new drugable targets of retinal neovascularization

AbstractRetinal neovascular diseases such as retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR) and retinal vein occlusion (RVO) are major causes of visual impairment in western countries. Oxygen‐induced retinopathy (OIR) model is widely used model for studying these retinopathies. The purpose of our study was to characterize changes in protein levels during OIR model using by a recently developed technique, sequential window acquisition of all theoretical mass spectra (SWATH‐MS) proteomics. Samples from normoxic and OIR animals were collected in three time points; P13, P17 and P42 and analyzed with SWATH‐MS. Immunoblotting and immunohistochemistry from mouse and human PDR samples was used to confirm these results and address their relevance for human neovascular retinopathies. We performed the most extensive proteomic profiling of OIR to date and quantified almost 3000 unique proteins and their expression levels in control and OIR retinas. The methodology used in the study allowed also temporal profiling of OIR proteome. The most prominent group of proteins induced at the early hypoxic phase of OIR were crystallins, while angiogenesis related proteins such as Filamin A and B, Annexin 2 and Myosin 9 were upregulated at the peak of neovascularization. In addition, the upstream regulator analysis identified potentially upregulated enhancers of angiogenesis, among them TGF‐β1. As retinal neovascularization regresses later in OIR, majority of the changes seen in protein expression levels returned to normal at P42, but there was still evidence suggesting that proteins involved in neurotransmission stayed at reduced levels.

Treatment of Pulmonary Hypertension With Angiotensin II Receptor Blocker and Neprilysin Inhibitor Sacubitril/Valsartan.

Angiotensin II has been implicated in maladaptive right ventricular (RV) hypertrophy and fibrosis associated with pulmonary hypertension (PH). Natriuretic peptides decrease RV afterload by promoting pulmonary vasodilation and inhibiting vascular remodeling but are degraded by neprilysin. We hypothesized that angiotensin receptor blocker and neprilysin inhibitor, sacubitril/valsartan (Sac/Val, LCZ696), will attenuate PH and improve RV function by targeting both pulmonary vascular and RV remodeling. PH was induced in rats using the SU5416/hypoxia model (Su/Hx), followed by 6-week treatment with placebo, Sac/Val, or Val alone. There were 4 groups: CON-normoxic animals with placebo (n=18); PH-Su/Hx rats+placebo (n=34); PH+Sac/Val (N=24); and PH+Val (n=16). In animals with PH, treatment with Sac/Val but not Val resulted in significant reduction in RV pressure (mm Hg: PH: 62±4, PH+Sac/Val: 46±5), hypertrophy (RV/LV+S: PH: 0.74±0.06, PH+Sac/Val: 0.46±0.06), collagen content (µg/50 µg protein: PH: 8.2±0.3, PH+Sac/Val: 6.4±0.4), pressures and improvement in RVs (mm/s: PH: 31.2±1.8, PH+Sac/Val: 43.1±3.6) compared with placebo. This was associated with reduced pulmonary vascular wall thickness, increased lung levels of ANP (atrial natriuretic peptide), BNP (brain-type natriuretic peptide), and cGMP, and decreased plasma endothelin-1 compared with PH alone. Also, PH+Sac/Val animals had altered expression of PKC isozymes in RV tissue compared with PH alone. Sac/Val reduces pulmonary pressures, vascular remodeling, as well as RV hypertrophy in a rat model of PH and may be appropriate for treatment of pulmonary hypertension and RV dysfunction.

Prenatal hypoxemia alters microglial morphology in fetal sheep

ObjectiveNeuroimmune cells, particularly microglia and astrocytes, play a critical role in neurodevelopment. Neurocognitive delays are common in children with congenital heart disease, but their etiology is poorly understood. Our objective was to determine whether prenatal hypoxemia, at levels common in congenital heart disease, induced neuroimmune activation to better understand the origins of neurobehavioral disorders in congenital heart disease. MethodsEight fetal sheep at gestational age 109 ± 3 days (term ∼145 days) were cannulated onto a pumpless extracorporeal oxygenator via the umbilical vessels and supported in a fluid environment for 22 ± 2 days under normoxic (n = 4) or hypoxic (n = 4) conditions. Control fetuses (n = 7) were harvested at gestational age 133 ± 4 days. At necropsy, brains were stained with ionized calcium-binding adaptor molecule 1 and glial fibrillary acidic protein antibodies to quantify microglia and astrocytes, respectively, in gray and white matter in frontotemporal and cerebellar sections. Microglia were classified into 4 morphologic types based on cell shape. Data were analyzed with 1-way analysis of variance or Fisher exact test, as appropriate. ResultsOxygen delivery was significantly reduced in hypoxic fetuses (15.6 ± 1.8 mL/kg/min vs 24.3 ± 2.3 mL/kg/min; P < .01). Rates of apoptosis were similar in hypoxic, normoxic, and intrauterine control animals in all examined areas. There were also no differences between groups in area occupied by glial fibrillary acidic protein-labeled astrocytes or ionized calcium-binding adaptor molecule 1-labeled microglia in all examined areas. However, round microglia were significantly increased in hypoxic animals compared with normoxic animals (33% vs 6%; P < .01) and control animals (33% vs 11%; P < .01). ConclusionsPrenatal hypoxemia altered microglial morphology without significant gliosis. Additional studies characterizing these mechanisms may provide insight into the origins of neurobehavioral disabilities in children with congenital heart disease.

Assessing the effect of hypoxia on cardiac metabolism using hyperpolarized 13 C magnetic resonance spectroscopy.

Hypoxia plays a role in many diseases and can have a wide range of effects on cardiac metabolism depending on the extent of the hypoxic insult. Noninvasive imaging methods could shed valuable light on the metabolic effects of hypoxia on the heart in vivo. Hyperpolarized carbon‐13 magnetic resonance spectroscopy (HP 13C MRS) in particular is an exciting technique for imaging metabolism that could provide such information.The aim of our work was, therefore, to establish whether hyperpolarized 13C MRS can be used to assess the in vivo heart's metabolism of pyruvate in response to systemic acute and chronic hypoxic exposure.Groups of healthy male Wistar rats were exposed to either acute (30 minutes), 1 week or 3 weeks of hypoxia. In vivo MRS of hyperpolarized [1‐13C] pyruvate was carried out along with assessments of physiological parameters and ejection fraction. Hematocrit was elevated after 1 week and 3 weeks of hypoxia.30 minutes of hypoxia resulted in a significant reduction in pyruvate dehydrogenase (PDH) flux, whereas 1 or 3 weeks of hypoxia resulted in a PDH flux that was not different to normoxic animals. Conversion of hyperpolarized [1‐13C] pyruvate into [1‐13C] lactate was elevated following acute hypoxia, suggestive of enhanced anaerobic glycolysis. Elevated HP pyruvate to lactate conversion was also seen at the one week timepoint, in concert with an increase in lactate dehydrogenase (LDH) expression. Following three weeks of hypoxic exposure, cardiac metabolism of pyruvate was comparable with that observed in normoxia.We have successfully visualized the effects of systemic hypoxia on cardiac metabolism of pyruvate using hyperpolarized 13C MRS, with differences observed following 30 minutes and 1 week of hypoxia. This demonstrates the potential of in vivo hyperpolarized 13C MRS data for assessing the cardiometabolic effects of hypoxia in disease.