Sostdc1 is expressed in all major compartments of developing and adult mammalian eyes.

PURPOSE: This study was conducted in order to study Sostdc1 expression in rat and human developing and adult eyes. METHODS: Using the yeast signal sequence trap screening method, we identified the Sostdc1 cDNA encoding a protein secreted by the adult rat retinal pigment epithelium. We determined by in situ hybridization, RT-PCR, immunohistochemistry, and western blot analysis Sostdc1 gene and protein expression in developing and postnatal rat ocular tissue sections. We also investigated Sostdc1 immunohistolocalization in developing and adult human ocular tissues. RESULTS: We demonstrated a prominent Sostdc1 gene expression in the developing rat central nervous system (CNS) and eyes at early developmental stages from E10.5 days postconception (dpc) to E13 dpc. Specific Sostdc1 immunostaining was also detected in most adult cells of rat ocular tissue sections. We also identified the rat ocular embryonic compartments characterized by a specific Sostdc1 immunohistostaining and specific Pax6, Sox2, Otx2, and Vsx2 immunohistostaining from embryonic stages E10.5 to E13 dpc. Furthermore, we determined the localization of SOSTDC1 immunoreactivity in ocular tissue sections of developing and adult human eyes. Indeed, we detected SOSTDC1 immunostaining in developing and adult human retinal pigment epithelium (RPE) and neural retina (NR) as well as in several developing and adult human ocular compartments, including the walls of choroidal and scleral vessels. Of utmost importance, we observed a strong SOSTDC1 expression in a pathological ocular specimen of type 2 Peters' anomaly complicated by retinal neovascularization as well in the walls ofother pathological extra-ocular vessels.  CONCLUSION: As rat Sostdc1 and human SOSTDC1 are dual antagonists of the Wnt/ß-catenin and BMP signaling pathways, these results underscore the potential crucial roles of these pathways and their antagonists, such as Sostdc1 and SOSTDC1, in developing and adult mammalian normal eyes as well as in syndromic and nonsyndromic congenital eye diseases.

ER Stress is Involved in Epithelial-To-Mesenchymal Transition of Alveolar Epithelial Cells Exposed to a Hypoxic Microenvironment.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal interstitial lung disease of unknown origin. Alveolar epithelial cells (AECs) play an important role in the fibrotic process as they undergo sustained endoplasmic reticulum (ER) stress, and may acquire a mesenchymal phenotype through epithelial-to-mesenchymal transition (EMT), two phenomena that could be induced by localized alveolar hypoxia. Here we investigated the potential links between hypoxia, ER stress and EMT in AECs. METHODS: ER stress and EMT markers were assessed by immunohistochemistry, western blot and qPCR analysis, both in vivo in rat lungs exposed to normoxia or hypoxia (equivalent to 8% O2) for 48 h, and in vitro in primary rat AECs exposed to normoxia or hypoxia (1.5% O2) for 2⁻6 days. RESULTS: Hypoxia induced expression of mesenchymal markers, pro-EMT transcription factors, and the activation of ER stress markers both in vivo in rat lungs, and in vitro in AECs. In vitro, pharmacological inhibition of ER stress by 4-PBA limited hypoxia-induced EMT. Calcium chelation or hypoxia-inducible factor (HIF) inhibition also prevented EMT induction under hypoxic condition. CONCLUSIONS: Hypoxia and intracellular calcium are both involved in EMT induction of AECs, mainly through the activation of ER stress and HIF signaling pathways.

Mesenchymal stem cells protect from hypoxia-induced alveolar epithelial-mesenchymal transition.

Administration of bone marrow-derived human mesenchymal stem cells (hMSC) reduces lung inflammation, fibrosis, and mortality in animal models of lung injury, by a mechanism not completely understood. We investigated whether hMSC would prevent epithelial-mesenchymal transition (EMT) induced by hypoxia in primary rat alveolar epithelial cell (AEC). In AEC cultured on semipermeable filters, prolonged hypoxic exposure (1.5% O2 for up to 12 days) induced phenotypic changes consistent with EMT, i.e., a change in cell morphology, a decrease in transepithelial resistance (Rte) and in the expression of epithelial markers [zonula occludens-1 (ZO-1), E-cadherin, AQP-5, TTF-1], together with an increase in mesenchymal markers [vimentin, α-smooth muscle actin (α-SMA)]. Expression of transcription factors driving EMT such as SNAIL1, ZEB1, and TWIST1 increased after 2, 24, and 48 h of hypoxia, respectively. Hypoxia also induced TGF-ß1 mRNA expression and the secretion of active TGF-ß1 in apical medium, and the expression of connective tissue growth factor (CTGF), two inducers of EMT. Coculture of AEC with hMSC partially prevented the decrease in Rte and in ZO-1, E-cadherin, and TTF-1 expression, and the increase in vimentin expression induced by hypoxia. It also abolished the increase in TGF-ß1 expression and in TGF-ß1-induced genes ZEB1, TWIST1, and CTGF. Finally, incubation with human recombinant KGF at a concentration similar to what was measured in hMSC-conditioned media restored the expression of TTF-1 and prevented the increase in TWIST1, TGF-ß1, and CTGF in hypoxic AEC. Our results indicate that hMSC prevent hypoxia-induced alveolar EMT through the paracrine modulation of EMT signaling pathways and suggest that this effect is partly mediated by KGF.

Comparative ventilatory strategies of acclimated rats and burrowing plateau pika (Ochotona curzoniae) in response to hypoxic-hypercapnia.

The objective of this study was to compare the different ventilatory strategies that help in coping with hypoxic-hypercapnia environment among two species: use acclimated rats and plateau pikas (Ochotona curzoniae) that live in Tibetan plateaus, and have been well adjusted to high altitude. Arterial blood samples taken at 4100 m of elevation in acclimatized rats and adapted pikas revealed inter-species differences with lower hemoglobin and hematocrit and higher blood pH in pikas. A linear and significant increase in minute ventilation was observed in pikas, which help them to cope with hypoxic-hypercapnia. Pikas also displayed a high inspiratory drive and an invariant respiratory timing regardless of the conditions. Biochemical analysis revealed that N-methyl-D-aspartate receptor (NMDA) receptor gene and nNOS gene are highly conserved between rats and pikas, however pikas have higher expression of NMDA receptors and nNOS compared to rats at the brainstem level. Taken together, these results suggest that pikas have developed a specific ventilatory pattern supported by a modification of the NMDA/NO ventilatory central pathways to survive in extreme conditions imposed on the Tibetan plateaus. These physiological adaptive strategies help in maintaining a better blood oxygenation despite high CO2 concentration in burrows at high altitude.

Oxygen modulates the glutathione peroxidase activity during the L6 myoblast early differentiation process.

AIM: This work aims to study the regulation of the glutathione peroxidase and catalase activities in myoblasts from the L6 line exposed to 21%, 5% and 1% O2 during the cell differentiation. MATERIAL AND METHODS: Rat L6 myoblasts were grown in 1%, 5% or 21% O2 in the presence or absence of N-acetyl cysteine. The cell proliferation was evaluated by determining the doubling time and kinetics of cultures by counting cells. The cell differentiation was analyzed by determining the myogenic fusion index using antibodies against the myosin heavy chain. The glutathione peroxidase and catalase activities were assayed. The p110-PI3K/Thr308-Akt pathway was studied using western blotting. The oxidative status of the cells was carried out by determining TBARS. RESULTS: 5% O2 improves the glutathione peroxidase activity, p110-PI3K/Thr308-Akt pathway and differentiation while 1% O2 alters all these parameters compared to 21% O2. NAC (0.5 mM) can prevent the deleterious effects of hypoxia (1% O2) on the L6 myoblast proliferation and enhances the myoblast differentiation when exposed to 21% O2. TBARS are reduced in 5% O2 compared to both 21% and 1% O2. CONCLUSION: The glutathione peroxidase activity and p110-PI3K/Thr308-Akt are both modulated in the same way by oxygen.

Chronic pulmonary fibrosis alters the functioning of the respiratory neural network.

Some patients with idiopathic pulmonary fibrosis present impaired ventilatory variables characterised by low forced vital capacity values associated with an increase in respiratory rate and a decrease in tidal volume which could be related to the increased pulmonary stiffness. The lung stiffness observed in pulmonary fibrosis may also have an effect on the functioning of the brainstem respiratory neural network, which could ultimately reinforce or accentuate ventilatory alterations. To this end, we sought to uncover the consequences of pulmonary fibrosis on ventilatory variables and how the modification of pulmonary rigidity could influence the functioning of the respiratory neuronal network. In a mouse model of pulmonary fibrosis obtained by 6 repeated intratracheal instillations of bleomycin (BLM), we first observed an increase in minute ventilation characterised by an increase in respiratory rate and tidal volume, a desaturation and a decrease in lung compliance. The changes in these ventilatory variables were correlated with the severity of the lung injury. The impact of lung fibrosis was also evaluated on the functioning of the medullary areas involved in the elaboration of the central respiratory drive. Thus, BLM-induced pulmonary fibrosis led to a change in the long-term activity of the medullary neuronal respiratory network, especially at the level of the nucleus of the solitary tract, the first central relay of the peripheral afferents, and the Pre-Bötzinger complex, the inspiratory rhythm generator. Our results showed that pulmonary fibrosis induced modifications not only of pulmonary architecture but also of central control of the respiratory neural network.

Acetazolamide and chronic hypoxia: effects on haemorheology and pulmonary haemodynamics.

We tested the effect of acetazolamide on blood mechanical properties and pulmonary vascular resistance (PVR) during chronic hypoxia. Six groups of rats were either treated or not treated with acetazolamide (curative: treated after 10 days of hypoxic exposure; preventive: treated before hypoxic exposure with 40 mg · kg(-1) · day(-1)) and either exposed or not exposed to 3 weeks of hypoxia (at altitude >5,500 m). They were then used to assess the role of acetazolamide on pulmonary artery pressure, cardiac output, blood volume, haematological and haemorheological parameters. Chronic hypoxia increased haematocrit, blood viscosity and PVR, and decreased cardiac output. Acetazolamide treatment in hypoxic rats decreased haematocrit (curative by -10% and preventive by -11%), PVR (curative by -36% and preventive by -49%) and right ventricular hypertrophy (preventive -20%), and increased cardiac output (curative by +60% and preventive by +115%). Blood viscosity was significantly decreased after curative acetazolamide treatment (-16%) and was correlated with PVR (r=0.87, p<0.05), suggesting that blood viscosity could influence pulmonary haemodynamics. The fall in pulmonary vascular hindrance (curative by -27% and preventive by -45%) after treatment suggests that acetazolamide could decrease pulmonary vessels remodelling under chronic hypoxia. The effect of acetazolamide is multifactorial by acting on erythropoiesis, pulmonary circulation, haemorheological properties and cardiac output, and could represent a pertinent treatment of chronic mountain sickness.

In Transgenic Erythropoietin Deficient Mice, an Increase in Respiratory Response to Hypercapnia Parallels Abnormal Distribution of CO2/H+-Activated Cells in the Medulla Oblongata.

Erythropoietin (Epo) and its receptor are expressed in central respiratory areas. We hypothesized that chronic Epo deficiency alters functioning of central respiratory areas and thus the respiratory adaptation to hypercapnia. The hypercapnic ventilatory response (HcVR) was evaluated by whole body plethysmography in wild type (WT) and Epo deficient (Epo-TAgh) adult male mice under 4%CO2. Epo-TAgh mice showed a larger HcVR than WT mice because of an increase in both respiratory frequency and tidal volume, whereas WT mice only increased their tidal volume. A functional histological approach revealed changes in CO2/H+-activated cells between Epo-TAgh and WT mice. First, Epo-TAgh mice showed a smaller increase under hypercapnia in c-FOS-positive number of cells in the retrotrapezoid nucleus/parafacial respiratory group than WT, and this, independently of changes in the number of PHOX2B-expressing cells. Second, we did not observe in Epo-TAgh mice the hypercapnic increase in c-FOS-positive number of cells in the nucleus of the solitary tract present in WT mice. Finally, whereas hypercapnia did not induce an increase in the c-FOS-positive number of cells in medullary raphe nuclei in WT mice, chronic Epo deficiency leads to raphe pallidus and magnus nuclei activation by hyperacpnia, with a significant part of c-FOS positive cells displaying an immunoreactivity for serotonin in the raphe pallidus nucleus. All of these results suggest that chronic Epo-deficiency affects both the pattern of ventilatory response to hypercapnia and associated medullary respiratory network at adult stage with an increase in the sensitivity of 5-HT and non-5-HT neurons of the raphe medullary nuclei leading to stimulation of f R for moderate level of CO2.

A New Model of Acute Exacerbation of Experimental Pulmonary Fibrosis in Mice.

RATIONALE: idiopathic pulmonary fibrosis (IPF) is the most severe form of fibrosing interstitial lung disease, characterized by progressive respiratory failure leading to death. IPF's natural history is heterogeneous, and its progression unpredictable. Most patients develop a progressive decline of respiratory function over years; some remain stable, but others present a fast-respiratory deterioration without identifiable cause, classified as acute exacerbation (AE). OBJECTIVES: to develop and characterize an experimental mice model of lung fibrosis AE, mimicking IPF-AE at the functional, histopathological, cellular and molecular levels. METHODS: we established in C57BL/6 male mice a chronic pulmonary fibrosis using a repetitive low-dose bleomycin (BLM) intratracheal (IT) instillation regimen (four instillations of BLM every 2 weeks), followed by two IT instillations of a simple or double-dose BLM challenge to induce AE. Clinical follow-up and histological and molecular analyses were done for fibrotic and inflammatory lung remodeling analysis. MEASUREMENTS AND MAIN RESULTS: as compared with a low-dose BLM regimen, this AE model induced a late burst of animal mortality, worsened lung fibrosis and remodeling, and superadded histopathological features as observed in humans IPF-AE. This was associated with stronger inflammation, increased macrophage infiltration of lung tissue and increased levels of pro-inflammatory cytokines in lung homogenates. Finally, it induced in the remodeled lung a diffuse expression of hypoxia-inducible factor 1α, a hallmark of tissular hypoxia response and a major player in the progression of IPF. CONCLUSION: this new model is a promising model of AE in chronic pulmonary fibrosis that could be relevant to mimic IPF-AE in preclinical trials.

Effect of exercise training in rats exposed to chronic hypoxia: Application for Monge's disease.

Physical exercise may improve hematological conditions in high altitude dwellers suffering from Chronic Mountain Sickness (CMS), in reducing hemoglobin concentration. Therefore, the present study aimed to characterize the effects of 1-month exercise training session in a model of rats exposed to chronic hypoxia. Four groups of male rats were studied: normoxic sedentary (NS, n = 8), normoxic training (NT, n = 8), hypoxic sedentary (HS, n = 8), and hypoxic training group (HT, n = 8). Hypoxic groups were exposed to hypobaric hypoxia for one month (PB =433 Torr). Training intensity was progressively increased from a running speed of 10.4 to 17.8 m/min. Chronic hypoxia led to an increase in hematocrit (HCT) associated with a decrease in plasma volume despite an increase in water intake. Training led to a reduction in HCT (p < 0.01), with a non-significant increase in plasma volume and weight gain. Hypoxia and training had inhibitory effects on haptoglobin (NS group: 379 ± 92; HT: 239 ± 34 µg/ml, p < 0.01). Chronic hypoxia and exercise training increased SpO2 measured after acute hypoxic exposure. Training blunted the decrease in V˙ O2 peak, time of exhaustion, and maximum speed associated with chronic exposure to hypoxia. Chronic hypoxia led to a right ventricular hypertrophy, which was not corrected by 1-month exercise training. Altogether, by decreasing hematocrit, reducing body weight, and limiting performance decrease, training in hypoxia may have a beneficial effect on excessive erythropoiesis in chronic hypoxia. Therefore, regular exercise training might be beneficial to avoid worsening of CMS symptoms in high altitude dwellers and to improve their quality of life.

Cytoprotective effects of erythropoietin: What about the lung?

Erythropoietin (Epo) is a pleiotropic cytokine, essential for erythropoiesis. Epo and its receptor (Epo-R) are produced by several tissues and it is now admitted that Epo displays other physiological functions than red blood cell synthesis. Indeed, Epo provides cytoprotective effects, which consist in prevention or fight against pathological processes. This perspective article reviews the various protective effects of Epo in several organs and tries to give a proof of concept about its effects in the lung. The tissue-protective effects of Epo could be a promising approach to limit the symptoms of acute and chronic lung diseases.

Sleep Apnea in Idiopathic Pulmonary Fibrosis: A Molecular Investigation in an Experimental Model of Fibrosis and Intermittent Hypoxia.

BACKGROUND: High prevalence of obstructive sleep apnea (OSA) is reported in incident and prevalent forms of idiopathic pulmonary fibrosis (IPF). We previously reported that Intermittent Hypoxia (IH), the major pathogenic element of OSA, worsens experimental lung fibrosis. Our objective was to investigate the molecular mechanisms involved. METHODS: Impact of IH was evaluated on C57BL/6J mice developing lung fibrosis after intratracheal instillation of Bleomycin (BLM). Mice were Pre-exposed 14 days to IH before induction of lung fibrosis or Co-challenged with IH and BLM for 14 days. Weight loss and survival were daily monitored. After experimentations, lungs were sampled for histology, and protein and RNA were extracted. RESULTS: Co-challenge or Pre-exposure of IH and BLM induced weight loss, increased tissue injury and collagen deposition, and pro-fibrotic markers. Major worsening effects of IH exposure on lung fibrosis were observed when mice were Pre-exposed to IH before developing lung fibrosis with a strong increase in sXBP1 and ATF6N ER stress markers. CONCLUSION: Our results showed that IH exacerbates BLM-induced lung fibrosis more markedly when IH precedes lung fibrosis induction, and that this is associated with an enhancement of ER stress markers.

Skeletal muscle intrinsic functional properties are preserved in a model of erythropoietin deficient mice exposed to hypoxia.

Erythropoietin (Epo)-induced polycythemia is the main factor of adaptation to hypoxia. In this study, we analysed the effects of Epo deficiency on intrinsic functional properties of slow and fast twitch muscles in a model of erythropoietin deficient mice (Epo-TAg(h)) exposed to hypoxia. We hypothesised that Epo deficiency would be deleterious for skeletal muscle structure and phenotype, which could change its functional properties and alters the adaptive response to ambient hypoxia. Wild-type (WT) and Epo-TAg(h) mice were left in hypobaric chamber at 420 mm Hg pressure for 14 days. Soleus (SOL) and extensor digitorum longus (EDL) were analysed in vitro by mechanical measurements, immunohistological and biochemical analyses. The results were compared to those obtained in corresponding muscles of age-matched normoxic groups. Our data did not show any difference between the groups whatever the Epo deficiency and/or hypoxic conditions for twitch force, tetanic force, fatigue, typology and myosin heavy chain composition. Normoxic Epo-TAg(h) mice exhibit improved capillary-to-fibre ratio compared to WT mice in both SOL and EDL whereas no angiogenic effects of hypoxia or combined Epo-deficiency/hypoxia were observed. These results suggest that skeletal muscles possess a great capacity of adaptation to Epo deficiency. Then Epo deficiency is not a sufficient factor to modify intrinsic functional properties of skeletal muscles.

Epo is relevant neither for microvascular formation nor for the new formation and maintenance of mice skeletal muscle fibres in both normoxia and hypoxia.

Erythropoietin (Epo) and vascular growth factor (VEGF) are known to be involved in the regulation of cellular activity when oxygen transport is reduced as in anaemia or hypoxic conditions. Because it has been suggested that Epo could play a role in skeletal muscle development, regeneration, and angiogenesis, we aimed to assess Epo deficiency in both normoxia and hypoxia by using an Epo-deficient transgenic mouse model (Epo-TAg(h)). Histoimmunology, ELISA and real time RT-PCR did not show any muscle fiber atrophy or accumulation of active HIF-1alpha but an improvement of microvessel network and an upregulation of VEGFR2 mRNA in Epo-deficient gastrocnemius compared with Wild-Type one. In hypoxia, both models exhibit an upregulation of VEGF120 and VEGFR2 mRNA but no accumulation of Epo protein. EpoR mRNA is not up-regulated in both Epo-deficient and hypoxic gastrocnemius. These results suggest that muscle deconditioning observed in patients suffering from renal failure is not due to Epo deficiency.

Long-term ventilatory adaptation and ventilatory response to hypoxia in plateau pika (Ochotona curzoniae): role of nNOS and dopamine.

We assessed ventilatory patterns and ventilatory responses to hypoxia (HVR) in high-altitude (HA) plateau pikas, repetitively exposed to hypoxic burrows, and control rats. We evaluated the role of neuronal nitric oxide synthase (nNOS) and dopamine by using S-methyl-l-thiocitrulline (SMTC) inhibitor and haloperidol antagonist, respectively. Ventilation (Vi) was measured using a whole body plethysmograph in conscious pikas (n = 9) and low-altitude (LA) rats (n = 7) at different Pi(O(2)) (56, 80, 111, 150, and 186 mmHg) and in HA acclimatized rats (n = 9, 8 days at 4,600 m) at two different Pi(O(2)) (56 and 80 mmHg). The effects of NaCl, SMTC, and haloperidol on ventilatory patterns were assessed in pikas at Pi(O(2)) = 56 and 80 mmHg. We observed a main species effect with larger Vi, tidal volume (VT), inspiratory time/total time (T(i)/T(tot)), and a lower expiratory time in pikas than in LA rats. Pikas had also a larger VT and lower respiratory frequency compared with HA rats in hypoxia. HVR of pikas and rats were not statistically different. In pikas, SMTC induced a significant increase in Vi and VT for a Pi(O(2)) of 56 mmHg, but had no effect for a PiO(2) of 80 mmHg, i.e., the living altitude of pikas. In pikas, haloperidol injection had no effect on any ventilatory parameter. Long-term ventilatory adaptation in pikas is mainly due to an improvement in respiratory pattern (VT and T(i)/T(tot)) with no significant improvement in HVR. The sensitivity to severe acute hypoxia in pikas seems to be regulated by a peripheral nNOS mechanism.

The Genetic Architecture of Chronic Mountain Sickness in Peru.

Chronic mountain sickness (CMS) is a pathological condition resulting from chronic exposure to high-altitude hypoxia. While its prevalence is high in native Andeans (>10%), little is known about the genetic architecture of this disease. Here, we performed the largest genome-wide association study (GWAS) of CMS (166 CMS patients and 146 controls living at 4,380 m in Peru) to detect genetic variants associated with CMS. We highlighted four new candidate loci, including the first CMS-associated variant reaching GWAS statistical significance (rs7304081; P = 4.58 × 10-9). By looking at differentially expressed genes between CMS patients and controls around these four loci, we suggested AEBP2, CAST, and MCTP2 as candidate CMS causal genes. None of the candidate loci were under strong natural selection, consistent with the observation that CMS affects fitness mainly after the reproductive years. Overall, our results reveal new insights on the genetic architecture of CMS and do not provide evidence that CMS-associated variants are linked to a strong ongoing adaptation to high altitude.

Modeling the Evans Blue Dilution Method for the Measurement of Plasma Volume in Small Animals: A New Optimized Method.

The measurement of plasma volume (Vp) in humans and animals is frequently performed by the Evans blue dye dilution method. However, after injection of Evans blue into the circulation, no steady state is observed because of delayed mixing and progressive leakage of dye out of vascular space. Various methods of calculation have been proposed, either with a single blood sampling 5-10 min after dye injection (Single point method), or with extrapolation at time zero of a logarithmic decay (Log linear method). We propose a method based on a two-compartment hypothesis taking into account the initial mixing and the leakage phase in the time course of dye concentration. Nineteen Sprague-Dawley rats were studied in various conditions and blood sampling was performed before and 2, 4 and 6 min after injection of 200 µg Evans blue. A mathematical model was designed to describe the two-compartment hypothesis and allowed the calculation of Vp and Kout (rate of disappearance of dye from vascular space). A Bland and Altman representation evidenced an overestimation of Vp with previous methods and the great dispersion of results with the single point method, especially when using the 6 min point. Calculation of Kout revealed more accurate with the model than the Log linear method, especially when the mixing rate is slow. We suggest using the two-compartment model to measure Vp with Evans blue technique in rats. This method also allows precise evaluation of the rate of dye leakage, which could be a good marker of vascular permeability to albumin.

HIF-1α triggers ER stress and CHOP-mediated apoptosis in alveolar epithelial cells, a key event in pulmonary fibrosis.

Endoplasmic Reticulum (ER) stress of alveolar epithelial cells (AECs) is recognized as a key event of cell dysfunction in pulmonary fibrosis (PF). However, the mechanisms leading to AECs ER stress and ensuing unfolded protein response (UPR) pathways in idiopathic PF (IPF) remain unclear. We hypothesized that alveolar hypoxic microenvironment would generate ER stress and AECs apoptosis through the hypoxia-inducible factor-1α (HIF-1α). Combining ex vivo, in vivo and in vitro experiments, we investigated the effects of hypoxia on the UPR pathways and ER stress-mediated apoptosis, and consecutively the mechanisms linking hypoxia, HIF-1α, UPR and apoptosis. HIF-1α and the pro-apoptotic ER stress marker C/EBP homologous protein (CHOP) were co-expressed in hyperplastic AECs from bleomycin-treated mice and IPF lungs, not in controls. Hypoxic exposure of rat lungs or primary rat AECs induced HIF-1α, CHOP and apoptosis markers expression. In primary AECs, hypoxia activated UPR pathways. Pharmacological ER stress inhibitors and pharmacological inhibition or silencing of HIF-1α both prevented hypoxia-induced upregulation of CHOP and apoptosis. Interestingly, overexpression of HIF-1α in normoxic AECs increased UPR pathways transcription factors activities, and CHOP expression. These results indicate that hypoxia and HIF-1α can trigger ER stress and CHOP-mediated apoptosis in AECs, suggesting their potential contribution to the development of IPF.

Intermittent Hypoxia Increases the Severity of Bleomycin-Induced Lung Injury in Mice.

BACKGROUND: Severe obstructive sleep apnea (OSA) with chronic intermittent hypoxia (IH) is common in idiopathic pulmonary fibrosis (IPF). Here, we evaluated the impact of IH on bleomycin- (BLM-) induced pulmonary fibrosis in mice. METHODS: C57BL/6J mice received intratracheal BLM or saline and were exposed to IH (40 cycles/hour; FiO2 nadir: 6%; 8 hours/day) or intermittent air (IA). In the four experimental groups, we evaluated (i) survival; (ii) alveolar inflammation, pulmonary edema, lung oxidative stress, and antioxidant enzymes; (iii) lung cell apoptosis; and (iv) pulmonary fibrosis. RESULTS: Survival at day 21 was lower in the BLM-IH group (p < 0.05). Pulmonary fibrosis was more severe at day 21 in BLM-IH mice, as assessed by lung collagen content (p = 0.02) and histology. At day 4, BLM-IH mice developed a more severe neutrophilic alveolitis, (p < 0.001). Lung oxidative stress was observed, and superoxide dismutase and glutathione peroxidase expression was decreased in BLM-IH mice (p < 0.05 versus BLM-IA group). At day 8, pulmonary edema was observed and lung cell apoptosis was increased in the BLM-IH group. CONCLUSION: These results show that exposure to chronic IH increases mortality, lung inflammation, and lung fibrosis in BLM-treated mice. This study raises the question of the worsening impact of severe OSA in IPF patients.

A large genomic deletion in the PDHX gene caused by the retrotranspositional insertion of a full-length LINE-1 element.

The long interspersed element-1 (LINE-1 or L1) retrotransposition has altered the human genome in many ways. In particular, recent in vitro studies have demonstrated that the retrotranspositional insertion of L1 elements has resulted in significant genomic deletions. Here we provide evidence for its operation in the human genome by identifying a approximately 46-kb pathological genomic deletion in the PDHX gene directly linked to the insertion of a full-length L1 element, in a patient with pyruvate dehydrogenase complex (PDHc) deficiency. Both the deduced bottom and top strand cleavage sites in the PDHX gene coincide with the consensus L1 endonuclease (EN) target sequence 5'-TTTT/A-3', while the full-length L1 element is followed by a 67-bp poly(A) tail. Interestingly, two hairpin structures, potentially formed by the inverted repeats present immediately 5' to the top strand nick site and 3' to the bottom strand nick site, may have facilitated the accessibility of L1 EN to the target sequences and also brought the two otherwise distantly located sequences into close proximity. Since the L1 element inserted in the PDHX gene is full-length, we favor the model of the template jumping as opposed to that of the microhomology-mediated end-joining for linking the 5' end of the nascent L1 copy to its genomic target. Our finding not only serves as an important complement to the in vitro approaches to studying L1 retrotransposition, but also reveals a novel mechanism causing human genetic disease.