Hypoxic Animals Research Articles

Alveolar Hypoxia Promotes Murine Lung Tumor Growth through a VEGFR-2/EGFR-Dependent Mechanism

Patients with chronic obstructive pulmonary disease (COPD) are at an increased risk for the development of lung cancer, the mechanisms for which are incompletely understood. We hypothesized that the hypoxic pulmonary microenvironment present in COPD would augment lung carcinogenesis. Mice were subjected to chemical carcinogenesis protocols and placed in either hypoxia or normoxia. Mice exposed to chronic hypoxia developed tumors with increased volume compared with normoxic controls. Both lungs and tumors from hypoxic mice showed a preferential stabilization of HIF-2α and increased expression of VEGF-A, FGF2, and their receptors as well as other survival, proliferation, and angiogenic signaling pathways regulated by HIF-2α. We showed that tumors arising in hypoxic animals have increased sensitivity to VEGFR-2/EGFR inhibition, as chemoprevention with vandetanib showed markedly increased activity in hypoxic mice. These studies showed that lung tumors arising in a hypoxic microenvironment express increased growth, angiogenic, and survival signaling that could contribute to the increased lung cancer risk in COPD. Furthermore, the differential sensitivity of tumors arising in hypoxia to VEGFR-2/EGFR inhibition suggests that the altered signaling present in tumors arising in hypoxic lung might be therapeutically exploited in patients with underlying COPD.

Infusing Sodium Bicarbonate Suppresses Hydrogen Peroxide Accumulation and Superoxide Dismutase Activity in Hypoxic-Reoxygenated Newborn Piglets

BackgroundThe effectiveness of sodium bicarbonate (SB) has recently been questioned although it is often used to correct metabolic acidosis of neonates. The aim of the present study was to examine its effect on hemodynamic changes and hydrogen peroxide (H2O2) generation in the resuscitation of hypoxic newborn animals with severe acidosis.MethodsNewborn piglets were block-randomized into a sham-operated control group without hypoxia (n = 6) and two hypoxia-reoxygenation groups (2 h normocapnic alveolar hypoxia followed by 4 h room-air reoxygenation, n = 8/group). At 10 min after reoxygenation, piglets were given either i.v. SB (2 mEq/kg), or saline (hypoxia-reoxygenation controls) in a blinded, randomized fashion. Hemodynamic data and blood gas were collected at specific time points and cerebral cortical H2O2 production was continuously monitored throughout experimental period. Plasma superoxide dismutase and catalase and brain tissue glutathione, superoxide dismutase, catalase, nitrotyrosine and lactate levels were assayed.ResultsTwo hours of normocapnic alveolar hypoxia caused cardiogenic shock with metabolic acidosis (pH: 6.99±0.07, HCO3 −: 8.5±1.6 mmol/L). Upon resuscitation, systemic hemodynamics immediately recovered and then gradually deteriorated with normalization of acid-base imbalance over 4 h of reoxygenation. SB administration significantly enhanced the recovery of both pH and HCO3− recovery within the first hour of reoxygenation but did not cause any significant effect in the acid-base at 4 h of reoxygenation and the temporal hemodynamic changes. SB administration significantly suppressed the increase in H2O2 accumulation in the brain with inhibition of superoxide dismutase, but not catalase, activity during hypoxia-reoxygenation as compared to those of saline-treated controls.ConclusionsDespite enhancing the normalization of acid-base imbalance, SB administration during resuscitation did not provide any beneficial effects on hemodynamic recovery in asphyxiated newborn piglets. SB treatment also reduced the H2O2 accumulation in the cerebral cortex without significant effects on oxidative stress markers presumably by suppressing superoxide dismutase but not catalase activity.

Chronic Hypoxia Suppresses Pregnancy-Induced Upregulation of Large-Conductance Ca 2+ -Activated K + Channel Activity in Uterine Arteries

Our previous study demonstrated that increased Ca(2+)-activated K(+) (BK(Ca)) channel activity played a key role in the normal adaptation of reduced myogenic tone of uterine arteries in pregnancy. The present study tested the hypothesis that chronic hypoxia during gestation inhibits pregnancy-induced upregulation of BK(Ca) channel function in uterine arteries. Resistance-sized uterine arteries were isolated from nonpregnant and near-term pregnant sheep maintained at sea level (≈ 300 m) or exposed to high-altitude (3801 m) hypoxia for 110 days. Hypoxia during gestation significantly inhibited pregnancy-induced upregulation of BK(Ca) channel activity and suppressed BK(Ca) channel current density in pregnant uterine arteries. This was mediated by a selective downregulation of BK(Ca) channel β1 subunit in the uterine arteries. In accordance, hypoxia abrogated the role of the BK(Ca) channel in regulating pressure-induced myogenic tone of uterine arteries that was significantly elevated in pregnant animals acclimatized to chronic hypoxia. In addition, hypoxia abolished the steroid hormone-mediated increase in the β1 subunit and BK(Ca) channel current density observed in nonpregnant uterine arteries. Although the activation of protein kinase C inhibited BK(Ca) channel current density in pregnant uterine arteries of normoxic sheep, this effect was ablated in the hypoxic animals. The results demonstrate that selectively targeting BK(Ca) channel β1 subunit plays a critical role in the maladaption of uteroplacental circulation caused by chronic hypoxia, which contributes to the increased incidence of preeclampsia and fetal intrauterine growth restriction associated with gestational hypoxia.

Effects of Hypoxia on Growth and Biomechanics in Limb Bones of Alligator mississippiensis

Microanatomy and cross sectional geometry of limb bones are often used to infer life history and locomotor ability of fossil vertebrates based on study of their extant relatives; often the extreme variations in atmospheric oxygen that occurred over geologic time (oxygen levels 30% or more during the Permian, 15% in the Triassic) are not considered. As hypoxia is known to inhibit growth in vertebrates, while hyperoxia may be permissive, such comparisons between extinct and modern taxa merit investigation. As members of Archosauria, a clade with a long evolutionary history, alligators are excellent models to test for effects of environmental oxygen on skeletal growth and biomechanics. We incubated eggs and reared hatchlings in hypoxic (16% O2) and hyperoxic (26%, 31%, 36% O2) conditions. After sacrifice, femora were used in whole‐bone morphometric measurements and preparation of transverse mid‐diaphyseal ground sections. For comparison, traits were standardized to femur length. Cross sectional area, second moment of area, and polar moment of inertia scaled with isometry in alligators reared in chronic hypoxia, but with negative allometry in hyperoxic groups. The stouter femora of hypoxic animals exhibited greater resistance to compression, bending and torsion than predicted. Caution in evaluation of paleohistological data and consideration of oxygen availability is suggested.Supported by NSF IOS‐0922756.

In vivo up-regulation of the unfolded protein response after hypoxia

BackgroundLow oxygen (O2) availability, a condition called hypoxia, has different and profound consequences in tissues and organs. Besides the hypoxia-inducible response, mammalian cells induce a coordinated cytoprotective pathway called Unfolded Protein Response (UPR). We studied the molecular basis of UPR and apoptosis in animal models exposed to different hypoxic stresses and assessed the ability of liver and myocardium to respond to low oxygen by activating different arms of the UPR according to the severity of the insults in a tissue specific manner. MethodsWe assessed the levels of several UPR markers in hypoxic animals by Real Time PCR and Western blotting. ResultsWhile the hepatocytes activate the apoptotic pathway mediated, in part, by CHOP and p-JNK, we could not detect an UPR-dependent apoptosis in myocytes. Moreover, severe hypoxia results in ATF4 translation, and induction of CHOP and GADD34 transcripts in liver, by contrast in the myocardium, the ATF4-CHOP-GADD34 signaling pathway is not detectably activated. General significanceComparison of several UPR markers in liver and myocardium enabled to underscore the ability of hepatocytes and myocites to selectively activate and fine tune the UPR signaling pathway during hypoxia in vivo.

17β-Estradiol Attenuates Hypoxic Pulmonary Hypertension via Estrogen Receptor–mediated Effects

17β-Estradiol (E2) attenuates hypoxic pulmonary vasoconstriction and hypoxic pulmonary hypertension (HPH) through an unknown mechanism that may involve estrogen receptors (ER) or E2 conversion to catecholestradiols and methoxyestradiols with previously unrecognized effects on cardiopulmonary vascular remodeling. To determine the mechanism by which E2 exerts protective effects in HPH. Male rats were exposed to hypobaric hypoxia while treated with E2 (75 μg/kg/d) or vehicle. Subgroups were cotreated with pharmacologic ER-antagonist or with inhibitors of E2-metabolite conversion. Complementary studies were performed in rats cotreated with selective ERα- or ERβ-antagonist. Hemodynamic and pulmonary artery (PA) and right ventricular (RV) remodeling parameters, including cell proliferation, cell cycle, and autophagy, were measured in vivo and in cultured primary rat PA endothelial cells. E2 significantly attenuated HPH endpoints. Hypoxia increased ERβ but not ERα lung vascular expression. Co-treatment with nonselective ER inhibitor or ERα-specific antagonist rendered hypoxic animals resistant to the beneficial effects of E2 on cardiopulmonary hemodynamics, whereas ERα- and ERβ-specific antagonists opposed the remodeling effects of E2. In contrast, inhibition of E2-metabolite conversion did not abolish E2 protection. E2-treated hypoxic animals exhibited reduced ERK1/2 activation and increased expression of cell-cycle inhibitor p27(Kip1) in lungs and RV, with up-regulation of lung autophagy. E2-induced signaling was recapitulated in hypoxic but not normoxic endothelial cells, and was associated with decreased vascular endothelial growth factor secretion and cell proliferation. E2 attenuates hemodynamic and remodeling parameters in HPH in an ER-dependent manner, through direct antiproliferative mechanisms on vascular cells, which may provide novel nonhormonal therapeutic targets for HPH.

Low-Dose Fluvastatin Reverses the Hypoxic Pulmonary Adventitial Fibroblast Phenotype in Experimental Pulmonary Hypertension

Hypoxic pulmonary hypertension is a worldwide public health problem. Statins attenuate hypoxic pulmonary hypertension in animal models, but the mechanism of action and applicability of these results to human treatment are not established. In hypoxic models, pulmonary artery fibroblast proliferation contributes substantially to pulmonary vascular remodeling. We previously showed that acute hypoxic pulmonary adventitial fibroblast proliferation can be selectively inhibited by statins and p38 mitogen-activated protein (MAP) kinase inhibitors. Here we used complementary chronic hypoxic and acute hypoxic coculture models to obtain necessary preclinical information regarding the utility of fluvastatin in the treatment of chronic hypoxic pulmonary hypertension. The effects of fluvastatin, cholesterol pathway intermediates, and related inhibitors on hypoxic adventitial fibroblast proliferation, p38 MAP kinase phosphorylation, and pulmonary artery smooth muscle cell proliferation were determined, using complementary chronic hypoxic rat and acute hypoxic bovine cell models. Fluvastatin reversed the proliferative phenotypic switch in adventitial fibroblasts from chronic hypoxic animals. This effect was circulation-specific, and implicated a Rac1-p38 MAP kinase signaling pathway. Coculture and conditioned media experiments also implicated this statin-sensitive signaling pathway in the release of pulmonary artery smooth muscle cell mitogens by hypoxic pulmonary adventitial fibroblasts. Treprostinil, sildenafil, and bosentan exerted no effect on the hypoxic fibroblast phenotype. Phenotypic changes (increased proliferation and mitogen release) in pulmonary artery fibroblasts during chronic hypoxia are dependent on a Rac1-p38 MAP kinase signaling pathway. The inhibition of these phenotypic changes with fluvastatin may be therapeutically relevant in high-altitude residents and in patients with hypoxic lung disease.

Activated CD47 promotes pulmonary arterial hypertension through targeting caveolin-1

Pulmonary arterial hypertension (PAH) is a progressive lung disease characterized by pulmonary vasoconstriction and vascular remodelling, leading to increased pulmonary vascular resistance and right heart failure. Loss of nitric oxide (NO) signalling and increased endothelial nitric oxide synthase (eNOS)-derived oxidative stress are central to the pathogenesis of PAH, yet the mechanisms involved remain incompletely determined. In this study, we investigated the role activated CD47 plays in promoting PAH. We report high-level expression of thrombospondin-1 (TSP1) and CD47 in the lungs of human subjects with PAH and increased expression of TSP1 and activated CD47 in experimental models of PAH, a finding matched in hypoxic human and murine pulmonary endothelial cells. In pulmonary endothelial cells CD47 constitutively associates with caveolin-1 (Cav-1). Conversely, in hypoxic animals and cell cultures activation of CD47 by TSP1 disrupts this constitutive interaction, promoting eNOS-dependent superoxide production, oxidative stress, and PAH. Hypoxic TSP1 null mice developed less right ventricular pressure and hypertrophy and markedly less arteriole muscularization compared with wild-type animals. Further, therapeutic blockade of CD47 activation in hypoxic pulmonary artery endothelial cells upregulated Cav-1, increased Cav-1CD47 co-association, decreased eNOS-derived superoxide, and protected animals from developing PAH. Activated CD47 is upregulated in experimental and human PAH and promotes disease by limiting Cav-1 inhibition of dysregulated eNOS.

Effect of ghrelin on aldolase gene expression in the heart of chronic hypoxic rat.

BackgroundChronic hypoxia causes apoptosis of cardiac myocytes, however, energy production by anaerobic glycolysis protects myocardium against hypoxia injuries. Aldolase A is a well-characterised key enzyme of the glycolysis pathway. Ghrelin, a 28-amino-acid peptide, synthesizes in the stomach and has protective roles in cardiovascular systems and also affects metabolic pathways.ObjectivesTherefore, the aim of this study was to evaluate the effect of ghrelin on aldolase A gene expression after chronic hypoxia in the rat hearts.Materials and MethodsTwenty four adult male wistar rats were randomly divided into three groups. Hypoxic rats with saline or ghrelin treatment were placed in a normobaric hypoxic chamber (O2 11 %), for two weeks. Controls remained in room air. Aldolase A gene expression was measured by Real-Time RT-PCR.Resultsthe transcriptiom rate of Aldolase A in hypoxic animals did not change significantly compared to negative control ones. During chronic hypoxia, ghrelin treatment increased the amount of heart Aldolase A gene expression compared to negative controls (P = 0.029). Hypoxic animals that were treated with ghrelin were significantly more polycythemic than the controls and even hypoxic with saline treated rats (P < 0.001).ConclusionsIt seems that ghrelin interferes in the cardiac metabolism through upregulation of glycolytic enzymes. In other words, it may protect heart from possible hypoxia induced damages.

Corticosterone synthesis inhibitor metyrapone ameliorates chronic hypobaric hypoxia induced memory impairment in rat

Chronic exposure to hypobaric hypoxia causes oxidative stress and neurodegeneration leading to memory impairment. The present study aimed at investigating the role of corticosterone in hypoxia induced neurodegeneration and effect of metyrapone, a corticosterone synthesis inhibitor that reduces the stress induced elevation of corticosterone without affecting the basal level, in ameliorating chronic hypobaric hypoxia induced cognitive decline. Rats were exposed to simulated altitude of 25,000 ft for 0, 3, 7, 14 and 21 days to determine the temporal alterations in corticosterone and its receptors following exposure to hypobaric hypoxia. Our results showed an elevation of corticosterone in plasma and hippocampal tissue following 7 days of exposure, which declined on prolonged hypoxic exposure for 21 days. A concomitant increase in ROS and lipid peroxidation was observed along with depletion of intracellular antioxidants. Glucocorticoid and mineralocorticoid receptors were upregulated on 3 and 7 days of hypoxic exposure. Though expression of Glut1 and Glut3 were upregulated on 3 days of hypoxic exposure, sharp decline in Glut1 expression following 7 days of hypoxic exposure leads to reduced neuronal glucose uptake. Administration of metyrapone from 3rd to 7th day of hypoxic exposure to suppress hypoxia induced increase in corticosterone levels resulted in reduced oxidative damage, neurodegeneration and improvement of intracellular energy status. The metyrapone treated hypoxic animals performed better in the Morris Water Maze. Further, administration of exogenous corticosterone along with metyrapone during hypoxic exposure blunted the neuroprotective effect of metyrapone indicating a role for corticosterone in mediating hypobaric hypoxia induced neurodegeneration and memory impairment.

Effect of ghrelin on protein kinase C-ε and protein kinase C-δ gene expression in the pulmonary arterial smooth muscles of chronic hypoxic rats.

Protein kinase C (PKC), can be activated in pulmonary arterial smooth muscle cells during hypoxia, leading to hypoxic pulmonary vasoconstriction (HPV). Studies are going on to detect the strict PKC isoform involved in the phenomenon. It has been shown that ghrelin, a 28-amino-acid peptide, may protect lungs from HPV side effects, to some extent. The aim of study was to evaluate the effect of exogenous ghrelin on PKC-ε and PKC-δ gene expression during chronic hypoxia. Twenty-four adult male Wistar rats were divided randomly in 3 groups. Hypoxic rats with saline or ghrelin treatment were placed in a normobaric hypoxic chamber for 2 weeks. Controls remained in room air. PKC-ε and PKC-δ gene expression was measured by real-time RT-PCR. Morphometric analysis showed that ghrelin reversed the hypoxia induced pulmonary artery wall thickness. In hypoxic animals, there was a 2- and 4-fold increment in PKC-ε and PKC- δ gene expression, respectively. Ghrelin treatment reduced the overexpression of PKC-ε and PKC-δ to control animals' value. Ghrelin by decreasing the expression of PKC-ε and PKC-δ in hypoxic animals reduces the HPV. Although more studies are needed, it could be an honest deduction that ghrelin affects HPV in a multifunctional manner and might be used as a therapeutic agent in the future.

Chronic hypoxia impairs extracellular nucleotide metabolism and barrier function in pulmonary artery vasa vasorum endothelial cells

Vascular remodeling plays a pivotal role in a variety of pathophysiological conditions where hypoxia and inflammation are prominent features. Intravascular ATP, ADP and adenosine are known as important regulators of vascular tone, permeability and homeostasis, however contribution of purinergic signalling to endothelial cell growth and angiogenesis remains poorly understood. By using vasa vasorum endothelial cells (VVEC) isolated from pulmonary artery adventitia of control and chronically hypoxic neonatal calves, these studies were aimed to evaluate the effect of hypoxia on biochemical and functional properties of microvascular endothelial network at the sites of angiogenesis. In comparison with normoxic controls, VVEC from hypoxic animals are characterized by (1) drastically impaired nucleoside triphosphate diphosphohydrolase-1 (NTPDase-1/CD39) and ecto-5'-nucleotidase/CD73 activities with respective increases in basal extracellular ATP and ADP levels (2) higher proliferative responses to low micromolar concentrations of ATP and ADP; and (3) enhanced permeability and disordered adenosinergic control of vascular barrier function (measured as a paracellular flux of 70 kDa fluorescein isothiocyanate-dextran). Together, these results suggest that unique pattern of purine-mediated angiogenic activation and enhanced leakiness of VVEC from chronically hypoxic vessels may be defined by disordered endothelial nucleotide homeostasis at sites of active neovascularization.

Inflammation contributes to the atherogenic role of intermittent hypoxia in apolipoprotein-E knock out mice

RationaleObstructive sleep apnea results in nocturnal intermittent hypoxia (IH) as a main trigger for cardiovascular morbidity, including atherosclerosis. IH induces hemodynamic, hormono-metabolic and also immuno-inflammatory alterations that could differentially contribute to atherosclerosis. Our study aimed at examining their respective contribution to the proatherogenic role of IH in atherosclerosis-prone mice. MethodsFifteen-week-old male apolipoprotein E-deficient (ApoE−/−) mice fed on a high-cholesterol diet (HCD) for 6 weeks and exposed for the last 14 days to IH (21–5% FiO2, 60s cycle, 8h/day) or air, were investigated for aortic atherosclerosis and lipid alterations. Then IH proatherogenicity was assessed in 15- and 20-week-old ApoE−/− mice fed on a standart-chow diet (SCD) exposed to IH or air for 14 days and assessed for atherosclerosis, lipid, hemodynamic and inflammation alterations. ResultsIH aggravated atherosclerosis in HCD-fed mice, whereas the extremely high cholesterol levels due to HCD were not different between normoxic and hypoxic animals. In SCD-fed mice, IH also aggravated atherosclerosis, more severely in 20 compared to 15-week-old animals. However, cholesterol levels that increased with IH were not different in the two SCD-fed groups. IH slightly elevated arterial blood pressure in 20-week-old animals only, and induced systemic and vascular inflammation, including increased splenocyte proliferation with decreased IL-10 secretion, and increased T-lymphocytes within atherosclerotic plaques. ConclusionsA short IH exposure without HCD has proatherogenic effects. In contrast to blood pressure or plasma lipids which were slightly or inconstantly affected by IH, inflammation at systemic and vascular levels appears as a potential contributing factor to IH atherogenicity.

Over-the-Wire Intravascular Ultrasound in an Animal Model of Pulmonary Hypertension

ABSTRACTBackground: Intravascular ultrasound (IVUS) measures prognostically important pulsatile flow indexes in patients with pulmonary hypertension (PH). IVUS catheters traditionally require a guiding catheter for placement which can impact hemodynamics in small infants because the guiding catheter renders the atrioventricular valve incompetent. Methods: Domestic swine (1.4–2.2 kg) were raised in isobaric normoxia (n = 4) or hypoxia (n = 3, FiO2 10–12%) for 72 hr for induction of PH. Cardiac catheterization and intravascular imaging was performed using a 3.5-Fr 20-MHz Eagle Eye Gold catheter (Volcano Corp., CA, USA) over a 0.014′′ guide wire. Intima-media thickness (IMT) was measured and relative area change and vascular pulsatility were calculated. Results: The IVUS probe was easily manipulated over a 0.014′′ wire without hemodynamic compromise in all animals. The IMT was thicker in the hypoxic group than the normoxic group (0.19 ± 0.03 mm vs. 0.31 ± 0.04 mm, p = .067). Hypoxic animals had systolic PH (39.66 ± 2.51 vs. 21.75 ± 2.87 mmHg, p = .02). Systemic arterial pressures between the groups were the same (hypoxic 68 ± 10.44 vs. normoxic 79.75 ± 14.84 mmHg, p = .26). Vascular pulsatility was similar (hypoxic 24 ± 2.64 vs. 20.25 ± 0.57%, p = .18). However, the arterial wall distensibility was significantly different (0.98 ± 0.2 vs. 2.01 ± 1.38 %/mmHg, p = .04). Conclusions: Monorail IVUS imaging without a guide catheter overcomes a major limitation for use in infants and small animal experimental models by avoiding hemodynamic compromise. This would be a valuable tool for assessment of PH in the research and clinical setting.

Tissue inhibitor of matrix metalloproteinase-1 mediates erythropoietin-induced neuroprotection in hypoxia ischemia

Previous studies have shown that erythropoietin (EPO) is neuroprotective in both in vivo and in vitro models of hypoxia ischemia. However these studies hold limited clinical translations because the underlying mechanism remains unclear and the key molecules involved in EPO-induced neuroprotection are still to be determined. This study investigated if tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and its upstream regulator signaling molecule Janus kinase-2 (JAK-2) are critical in EPO-induced neuroprotection. Hypoxia ischemia (HI) was modeled in-vitro by oxygen and glucose deprivation (OGD) and in-vivo by a modified version of Rice–Vannucci model of HI in 10-day-old rat pups. EPO treated cells were exposed to AG490, an inhibitor of JAK-2 or TIMP-1 neutralizing antibody for 2 h with OGD. Cell death, phosphorylation of JAK-2 and signal transducers and activators of transcription protein-3 (STAT-3), TIMP-1 expression, and matrix metalloproteinase-9 (MMP-9) activity were measured and compared with normoxic group. Hypoxic ischemic animals were treated one hour following HI and evaluated 48 h after. Our data showed that EPO significantly increased cell survival, associated with increased TIMP-1 activity, phosphorylation of JAK-2and STAT-3, and decreased MMP-9 activity in vivo and in vitro. EPO's protective effects were reversed by inhibition of JAK-2 or TIMP-1 in both models. We concluded that JAK-2, STAT-3 and TIMP-1 are key mediators of EPO-induced neuroprotection during hypoxia ischemia injury.

Bosentan attenuates right ventricular hypertrophy and fibrosis in normobaric hypoxia model of pulmonary hypertension

Maladaptive right ventricular (RV) hypertrophic responses lead to RV dysfunction and failure in patients with pulmonary arterial hypertension, but the mechanisms responsible for these changes are not well understood. The objective of this study was to evaluate the effect of treatment with bosentan on RV hypertrophy (RVH), fibrosis and expression of protein kinase C (PKC) isoforms in the RV of rats exposed to chronic hypoxia. Adult Sprague-Dawley rats were housed in normoxia or hypoxia (FIO(2) = 10%) and administered vehicle or 100 mg/kg/day bosentan. After 3 weeks, echocardiographic and hemodynamic assessment was performed. PKC, procollagen-1 and collagen expression levels were assessed using immunoblot or colorimetric assay. RV systolic pressure (RVSP) and RVH were higher in hypoxic compared with normoxic animals (RVSP: 72 ± 4 vs 25 ± 2 mm Hg, p < 0.05; RVH: 1.2 ± 0.06 vs 0.5 ± 0.03 mg/g body weight, p < 0.05). Bosentan had no effect on RVSP or mass in normoxic animals, but did attenuate RVH in hypoxic animals (hypoxic/vehicle: 1.2 ± 0.06; hypoxic/bosentan: 1.0 ± 0.05 mg/g body weight; p < 0.05). Hypoxia increased RV procollagen-1, and total collagen expression, effects that were attenuated by bosentan treatment. Hypoxia increased RV total and cytosolic PKC-δ protein expression, but had no effect on PKC-α or -ε isoforms. Administration with bosentan did not affect total PKC-δ protein expression. However, animals treated with bosentan had an increase in membranous PKC-δ when exposed to hypoxia. Bosentan inhibits RVH and RV collagen expression in rats exposed to chronic hypoxia, possibly via alteration of PKC-δ activity.

The effect of acidosis on pulmonary vascular smooth muscle cell metabolic response to hypoxia: implications for Pulmonary Hypertension

Background A glycolytic shift is key to apoptotic resistance in cancer cells and pulmonary vascular smooth muscle cells in pulmonary hypertension (PH). Its reversal may lead to apoptosis and reverse pulmonary vascular remodeling. We found that acidosis reversed remodeling in experimental PH and sought to investigate the metabolic response of pulmonary artery smooth muscle cells (PASMCs) to hypoxia. Materials and Methods We exposed rats to hypoxia to develop PH; acidosis was induced by ammonium chloride (NH4Cl) in drinking water. Primary PASMCs were exposed to acidosis (pH 6.8), physiologic pH (7.4) or hypoxia. Glucose Transporter-1 (Glut-1) mRNA and protein were assessed in lungs and PASMCs. Mitochondrial membrane potential (MMP) and apoptosis were assessed by JC-1 and caspase-3 cleavage, respectively. Results Glut-1 mRNA increased in lungs of hypoxic animals compared to normoxic with a decrease in hypoxic animals treated with NH4Cl. Isolated PASMCs showed higher Glut-1 mRNA in hypoxia than normoxia at physiologic and acidic pH. Acidic pH decreased Glut-1 protein in normoxia and hypoxia. Exposure to acidic pH decreased MMP and increased apoptosis by hydrogen peroxide. Conclusions Acidosis results in decreased Glut-1 in lungs and PASMCs, a decreased MMP and increased propensity to apoptosis. We speculate that acidosis reverses the glycolytic phenotype in hypoxia, thus promoting an apoptotic phenotype. Supported by NIH grant: 5T32HD007466

Acriflavine Attenuates the Development of Hypoxic Pulmonary Hypertension

Hypoxia Inducible Factor (HIF) is a highly conserved, heterodimeric transcription factor that has been shown to mediate the development of hypoxia pulmonary hypertension (HPH). Previous studies have identified acriflavine as a drug that binds directly to the PAS-B domain of HIF-1α and HIF-2 α inhibiting dimerization. Therefore, we hypothesized that treatment with acriflavine could reduce the development of HPH. In this study, adult male Wistar rats were weighed daily and injected with 2 mg/kg of acriflavine or saline, while being housed in either room air or in a chamber flushed with a mix of air and nitrogen to achieve 10% O2. After 21 days, we measured right ventricular pressure (RVP) via closed chest cardiac puncture, right ventricle and left ventricle plus septum weights and hematocrit (HCT). Pulmonary arterial smooth muscle cells were isolated from intrapulmonary arteries and used for measurement of intracellular calcium concentration [Ca2+]i using Fura-2 AM. In saline treated animals, chronic hypoxia lead to increased RVP, right ventricle hypertrophy, HCT, and [Ca2+]i. All of these variables were significantly reduced in chronically hypoxic animals receiving acriflavine. These results indicate that acriflavine attenuates the development of HPH.

HIF signaling and overall gene expression changes during hypoxia and prolonged exercise differ considerably

Exercise as well as hypoxia cause an increase in angiogenesis, changes in mitochondrial density and alterations in metabolism, but it is still under debate whether the hypoxia inducible factor (HIF) is active during both situations. In this study gene expression analysis of zebrafish larvae that were raised under normoxic, hypoxic, or training conditions were compared, using microarray analysis, quantitative real-time PCR and protein data. Although HIF expression is posttranslationally regulated, mRNA expression levels of all three isoforms (HIF-1α, HIF-2α, and HIF-3α) differed in each of the experimental groups, but the changes observed in hypoxic animals were much smaller than in trained larvae. Prominent changes were seen for Hif-2α expression, which significantly increased after the first day of exercise and then decreased down to values significantly below control values. HIF-3α mRNA expression in turn increased significantly, and at the end of the training period (9-15 days postfertilization) it was elevated three times. At the protein level a transient increase in HIF-1α was observed in hypoxic larvae, whereas in the exercise group the amount of HIF-1α protein even decreased below the level of control animals. The analyzed transcriptome was more affected in hypoxic zebrafish larvae, and hardly any genes were similarly altered by both treatments. These results clearly showed that HIF proteins played different roles in trained and hypoxic zebrafish larvae and that the exercise-induced transition to a more aerobic phenotype was not achieved by persistent activation of the hypoxic signaling pathway.

Hypoxia in early life is associated with lasting changes in left ventricular structure and function at maturity in the rat

BackgroundThere is a growing population of adults with repaired cyanotic congenital heart disease. These patients have increased risk of impaired cardiac health and premature death. We hypothesized that hypoxia in early life before surgical intervention causes lasting changes in left ventricular structure and function with physiological implications in later life. MethodsSprague–Dawley rats reared initially hypoxic conditions (FiO2=0.12) for days 1–10 of life were compared to rats reared only in ambient air. Cellular morphology and viability were compared among LV cardiomyocytes and histological analyses were performed on LV myocardium and arterioles. Intracellular calcium transients and cell shortening were measured in freshly-isolated cardiomyocytes, and mitochondrial hexokinase 2 (HK2) expression and activity were determined. Transthoracic echocardiography was used to assess LV function in anesthetized animals. ResultsCardiomyocytes from adult animals following hypoxia in early life had greater cellular volumes but significantly reduced viability. Echocardiographic analyses revealed LV hypertrophy and diastolic dysfunction, and alterations in cardiomyocyte calcium transients and cell shortening suggested impaired diastolic calcium reuptake. Histological analyses revealed significantly greater intima–media thickness and decreased lumen area in LV arterioles from hypoxic animals. Alterations in mitochondrial HK2 protein distribution and activity were also observed which may contribute to cardiomyocyte fragility. ConclusionsHypoxia in early life causes lasting changes in left ventricular structure and function that may negatively influence myocardial and vascular responses to physiological stress in later life. These data have implications for the growing population of adults with repaired or palliated cyanotic congenital heart disease.