Hypoxia-exposed Mice Research Articles

Lysyl oxidases play a causal role in vascular remodeling in clinical and experimental pulmonary arterial hypertension.

Pulmonary vascular remodeling, the pathological hallmark of pulmonary arterial hypertension, is attributed to proliferation, apoptosis resistance, and migration of vascular cells. A role of dysregulated matrix cross-linking and stability as a pathogenic mechanism has received little attention. We aimed to assess whether matrix cross-linking enzymes played a causal role in experimental pulmonary hypertension (PH). All 5 lysyl oxidases were detected in concentric and plexiform vascular lesions of patients with idiopathic pulmonary arterial hypertension. Lox, LoxL1, LoxL2, and LoxL4 expression was elevated in lungs of patients with idiopathic pulmonary arterial hypertension, whereas LoxL2 and LoxL3 expression was elevated in laser-capture microdissected vascular lesions. Lox expression was hypoxia-responsive in pulmonary artery smooth muscle cells and adventitial fibroblasts, whereas LoxL1 and LoxL2 expression was hypoxia-responsive in adventitial fibroblasts. Lox expression was increased in lungs from hypoxia-exposed mice and in lungs and pulmonary artery smooth muscle cells of monocrotaline-treated rats, which developed PH. Pulmonary hypertensive mice exhibited increased muscularization and perturbed matrix structures in vessel walls of small pulmonary arteries. Hypoxia exposure led to increased collagen cross-linking, by dihydroxylysinonorleucine and hydroxylysinonorleucine cross-links. Administration of the lysyl oxidase inhibitor β-aminopropionitrile attenuated the effect of hypoxia, limiting perturbations to right ventricular systolic pressure, right ventricular hypertrophy, and vessel muscularization and normalizing collagen cross-linking and vessel matrix architecture. Lysyl oxidases are dysregulated in clinical and experimental PH. Lysyl oxidases play a causal role in experimental PH and represent a candidate therapeutic target. Our proof-of-principle study demonstrated that modulation of lung matrix cross-linking can affect pulmonary vascular remodeling associated with PH.

The Nuclear Factor Erythroid 2–Related Factor 2 Activator Oltipraz Attenuates Chronic Hypoxia–Induced Cardiopulmonary Alterations in Mice

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator that activates many antioxidant enzymes. Oxidative stress, which accumulates in diseased lungs associated with pulmonary hypertension (PH), is thought to be responsible for the progression of cardiopulmonary changes. To test whether Nrf2 activation would exert therapeutic efficacy against cardiopulmonary changes in a hypoxia-induced PH model, wild-type (WT) and Nrf2-deficient mice as well as Kelch-like ECH associating protein 1 (Keap1) (negative regulator of Nrf2) knockdown mutant mice were exposed to hypobaric hypoxia for 3 weeks. This chronic hypoxia exacerbated right ventricular systolic pressure, right ventricular hypertrophy (RVH), and pulmonary vascular remodeling in the WT mice. These pathological changes were associated with aberrant accumulation of Tenascin-C, a disease-indicative extracellular glycoprotein. Simultaneous administration of oltipraz, a potent Nrf2 activator, significantly attenuated RVH and pulmonary vascular remodeling and concomitantly ameliorated Tenascin-C accumulation in the hypoxic mice. Hypoxia-exposed Nrf2-deficient mice developed more pronounced RVH than WT mice, whereas hypoxia-exposed Keap1-knockdown mice showed less RVH and pulmonary vascular remodeling than WT mice, underscoring the beneficial potency of Nrf2 activity against PH. We also demonstrated that expression of the Nrf2-regulated antioxidant enzymes was decreased in a patient with chronic obstructive pulmonary disease associated with PH. The decreased antioxidant enzymes may underlie the pathogenesis of cardiopulmonary changes in the patient with chronic obstructive pulmonary disease and PH. The pharmacologically or genetically induced Nrf2 activity clearly decreased RVH and pulmonary vascular remodeling in the hypoxic PH model. The efficacy of oltipraz highlights a promising therapeutic potency of Nrf2 activators for the prevention of PH in patients with hypoxemic lung disease.

Neurodevelopmental impairment following neonatal hyperoxia in the mouse

Extremely premature infants are often exposed to supra-physiologic concentrations of oxygen, and frequently have hypoxemic episodes. These preterm infants are at high risk (~40%) for neurodevelopmental impairment (NDI) even in the absence of obvious intracranial pathology such as intraventricular hemorrhage or periventricular leukomalacia. The etiology for NDI has not been determined, and there are no animal models to simulate neurodevelopmental outcomes of prematurity. Our objectives were to develop and characterize a mouse model to determine long-term effects of chronic hypoxia or hyperoxia exposure on neurodevelopment. Newborn C57BL/6 mice were exposed to hypoxia (12% O2) or hyperoxia (85% O2) from postnatal days 1 to 14 and then returned to air. At 12–14weeks of age, neurobehavioral assessment (Water Maze test, Novel Object Recognition test, Open Field test, Elevated Plus Maze, and Rotarod test) was performed, followed by MRI and brain histology. Neurobehavioral testing revealed that hyperoxia-exposed mice did poorly on the water maze and novel object recognition tests compared to air-exposed mice. MRI demonstrated smaller hippocampi in hyperoxia- and hypoxia-exposed mice with a greater reduction in hyperoxia-exposed mice, including a smaller cerebellum in hyperoxia-exposed mice. Brain histology showed reduced CA1 and CA3 and increased dentate gyral width in hippocampus. In conclusion, neonatal hyperoxia in mice leads to abnormal neurobehavior, primarily deficits in spatial and recognition memory, associated with smaller hippocampal sizes, similar to findings in ex-preterm infants. This animal model may be useful to determine mechanisms underlying developmental programming of NDI in preterm infants, and for evaluation of therapeutic strategies.

Intermittent hypoxia-activated cyclooxygenase pathway: role in atherosclerosis

Intermittent hypoxia, the main stimulus of obstructive sleep apnoea (OSA), induces inflammation, leading to early atherosclerosis. Whether the cyclooxygenase (COX) pathway contributes to intermittent hypoxia-induced atherosclerosis remains to be determined. We studied the effects of 8-weeks of intermittent hypoxia exposure on COX-pathway gene expression and atherosclerosis, and the influence of COX-1 inhibition by SC-560 on atherosclerosis progression in aortas of apolipoprotein E(-/-) mice. Urinary 11-dehydrothromboxane B2 (11-dTXB2) was assessed in 50 OSA subjects free of cardiovascular risk factor matched for age and body mass index with 25 controls, and 56 OSA with cardiovascular risk factor. Intermittent hypoxia significantly increased atherosclerotic lesion sizes, mRNA levels of COX-1 and thromboxane synthase (TXBS). Lesion sizes correlated to COX-1 (r = 0.654, p = 0.0003) and TXBS (r = 0.693, p<0.0001) mRNA levels. COX-1 inhibition reduced lesion progression in intermittent hypoxia mice only (p = 0.04). Urinary 11-dTXB2 was similar in OSA subjects free of cardiovascular risk factor and controls, but was increased by 13% (p = 0.007) in OSA subjects with cardiovascular risk factor compared with those without. Although OSA itself was not associated with increased urinary 11-dTXB2 concentration, the COX-1 pathway was activated in intermittent hypoxia-exposed mice and in OSA subjects presenting with cardiovascular risk factor, and may contribute to intermittent hypoxia-induced atherogenesis. COX-1 inhibition could be of clinical interest in the prevention of cardiovascular morbidity in OSA.

Bortezomib Alleviates Experimental Pulmonary Arterial Hypertension

Vascular remodeling and endothelial dysfunction are important pathogenic features of pulmonary arterial hypertension (PAH). There is a growing body of evidence that proteasome inhibitors may be beneficial in vascular diseases by inhibiting proliferation of vascular smooth muscle cells (VSMCs) and ameliorating endothelial dysfunction. Here, we evaluated whether bortezomib (BTZ) could alleviate hypoxia- and monocrotaline (MCT)-induced PAH. BTZ (at doses from 1 to 100 μg/kg, or a dose of 100 μg/kg) was administered to mice every other day for the last 2 weeks of a 5-week hypoxia (10% O(2)) period, or to rats once daily from Day 22 to Day 34 after MCT challenge, respectively. BTZ treatment substantially suppressed elevation of right ventricular (RV) systolic pressure, RV hypertrophy, and pulmonary vascular remodeling in hypoxia-exposed mice. Similarly, BTZ treatment inhibited RV hypertrophy and vascular remodeling in MCT-injected rats. Strikingly, BTZ rescued 70% of MCT-injected rats up to Day 60, along with a considerable reduction in RV systolic pressure and suppression of vascular remodeling, whereas, among MCT-injected rats not administered BTZ, there were no survivors by Day 41. BTZ significantly suppressed proliferation of pulmonary VSMCs in vivo and in vitro. Furthermore, BTZ increased not only endothelial nitric oxide (NO) synthase (eNOS), phosphorylated eNOS, and NO production in vitro, but also eNOS and p-eNOS in hypoxia-exposed mice and MCT-injected rats, respectively. In contrast to the beneficial effects, BTZ increased active caspase-3 in cardiac ventricles of MCT-injected rats. Taken together, with caution for cardiotoxicity, BTZ could be a potential therapeutic strategy in PAH, possibly acting by inhibition of VSMC proliferation and amelioration of endothelial dysfunction.

PAR-2 Inhibition Reverses Experimental Pulmonary Hypertension

A hallmark of the vascular remodeling process underlying pulmonary hypertension (PH) is the aberrant proliferation and migration of pulmonary arterial smooth muscle cells (PASMC). Accumulating evidence suggests that mast cell mediators play a role in the pathogenesis of PH. In the present study we investigated the importance of protease-activated receptor (PAR)-2 and its ligand mast cell tryptase in the development of PH. Our results revealed strong increase in PAR-2 and tryptase expression in the lungs of idiopathic pulmonary arterial hypertension (IPAH) patients, hypoxia-exposed mice, and monocrotaline (MCT)-treated rats. Elevated tryptase levels were also detected in plasma samples from IPAH patients. Hypoxia and platelet-derived growth factor (PDGF)-BB upregulated PAR-2 expression in PASMC. This effect was reversed by HIF (hypoxia inducible factor)-1α depletion, PDGF-BB neutralizing antibody, or the PDGF-BB receptor antagonist Imatinib. Attenuation of PAR-2 expression was also observed in smooth muscle cells of pulmonary vessels of mice exposed to hypoxia and rats challenged with MCT in response to Imatinib treatment. Tryptase induced PASMC proliferation and migration as well as enhanced synthesis of fibronectin and matrix metalloproteinase-2 in a PAR-2- and ERK1/2-dependent manner, suggesting that PAR-2-dependent signaling contributes to vascular remodeling by various mechanisms. Furthermore, PAR-2(-/-) mice were protected against hypoxia-induced PH, and PAR-2 antagonist application reversed established PH in the hypoxia mouse model. Our study identified a novel role of PAR-2 in vascular remodeling in the lung. Interference with this pathway may offer novel therapeutic options for the treatment of PH.

MiR-21 regulates chronic hypoxia-induced pulmonary vascular remodeling

Chronic hypoxia causes pulmonary vascular remodeling leading to pulmonary hypertension (PH) and right ventricle (RV) hypertrophy. Aberrant expression of microRNA (miRNA) is closely associated with a number of pathophysiologic processes. However, the role of miRNAs in chronic hypoxia-induced pulmonary vascular remodeling and PH has not been well characterized. In this study, we found increased expression of miR-21 in distal small arteries in the lungs of hypoxia-exposed mice. Putative miR-21 targets, including bone morphogenetic protein receptor (BMPR2), WWP1, SATB1, and YOD1, were downregulated in the lungs of hypoxia-exposed mice and in human pulmonary artery smooth muscle cells (PASMCs) overexpressing miR-21. We found that sequestration of miR-21, either before or after hypoxia exposure, diminished chronic hypoxia-induced PH and attenuated hypoxia-induced pulmonary vascular remodeling, likely through relieving the suppressed expression of miR-21 targets in the lungs of hypoxia-exposed mice. Overexpression of miR-21 enhanced, whereas downregulation of miR-21 diminished, the proliferation of human PASMCs in vitro and the expression of cell proliferation associated proteins, such as proliferating cell nuclear antigen, cyclin D1, and Bcl-xL. Our data suggest that miR-21 plays an important role in the pathogenesis of chronic hypoxia-induced pulmonary vascular remodeling and also suggest that miR-21 is a potential target for novel therapeutics to treat chronic hypoxia associated pulmonary diseases.

Origin of neomuscularized vessels in mice exposed to chronic hypoxia

Exposure of mice to chronic hypoxia is one of the most often used animal models to study pulmonary hypertension. Hypoxia exposure leads to vascular remodeling and muscularization of the small parenchymal vessels in the lung. Due to the anatomical differences between mice and humans, it is not possible to determine whether the remodeled vessels originate from the arterial or venous side of the vasculature. By applying antibodies against specific marker molecules expressed by arterial (ephrinB2) and venous (EphB4) endothelial cells, we could show that remodeled parenchymal vessels in hypoxia-exposed mice are mostly of arterial origin with slight venous involvement. Using these tools, it is possible to further characterize remodeled vessels in other small animal models, such as transgenic or knockout mice. Particularly useful applications would include selection of parenchymal vessels for laser microdissection studies.

Hypoxic preconditioning involves system Xc − regulation in mouse neural stem cells

In animals, hypoxic preconditioning has been used as a form of neuroprotection. The exact mechanism involved in neuroprotective hypoxic preconditioning has not been described, yet could be valuable for possible neuroprotective strategies. The overexpression of the cystine-glutamate exchanger, system Xc −, has been demonstrated as being neuroprotective (Shih, Erb et al. 2006). Here, using immunohistochemistry, we demonstrate that C57BL/6 mice exposed to hypoxia showed an increase in system Xc − expression, with the highest level of intensity in the hippocampus. Western Blot analysis also showed an almost 2-fold increase in system Xc − protein in hypoxia-exposed versus control mice. The mRNA for the regulatory subunit of system Xc −, xCT, and the xCT/actin ratio were also increased under hypoxic conditions. Experiments using hypoxia-inducible factor (HIF-1α) siRNA showed a statistically significant decrease in HIF-1α and system Xc − expression. Under hypoxic conditions, system Xc − activity, as determined by cystine uptake, increased 2-fold. Importantly, hypoxic preconditioning was attenuated in neural stem cells by pharmacological inhibition of system Xc − activity with S4-carboxyphenylglycine. These data provide the first evidence of hypoxic regulation of the cystine glutamate exchanger system Xc −.

Targeting of c-kit+ haematopoietic progenitor cells prevents hypoxic pulmonary hypertension

Haematopoietic c-kit+ progenitor cells may contribute to pulmonary vascular remodelling and pulmonary hypertension (PH). Stromal derived factor-1 (SDF-1/CXCL12) and its receptors CXCR4 and CXCR7 have been shown to be critical for homing and mobilisation of haematopoietic c-kit+ progenitor cells in the perivascular niche. We administered AMD3100, a CXCR4 antagonist, and CCX771, a CXCR7 antagonist, to chronic hypoxia exposed mice in order to study the role of c-kit+ progenitor cells in PH. CXCL12, CXCR4 and CXCR7 protein expression, haemodynamic parameters, right ventricular mass, extent of vascular remodelling and perivascular progenitor cell accumulation were studied. Chronic hypoxia-exposed mice showed increased total lung tissue expression of CXCR4, CXCR7 and CXCL12 after development of PH. This was associated with significantly increased right ventricular systolic pressure and evidence of right ventricular hypertrophy, vascular remodelling and perivascular c-kit+/sca-1+ progenitor cell accumulation. CCX771 administration did not abrogate these effects. In contrast, administration of AMD3100, whether alone or combined with CCX771, prevented vascular remodelling, PH and perivascular accumulation of c-kit+/sca-1+ progenitor cells, with a synergistic effect of these agents. This study offers important pathophysiological insights into the role of haematopoietic c-kit+ progenitors in hypoxia-induced vascular remodelling and may have therapeutic implications for PH.

Smurf1 ubiquitin ligase causes downregulation of BMP receptors and is induced in monocrotaline and hypoxia models of pulmonary arterial hypertension

Reduced bone morphogenetic protein (BMP) receptor (BMPR) expression and BMP signaling have been implicated in vascular cell proliferation and remodeling associated with pulmonary arterial hypertension (PAH). The low penetrance of the BMPR II disease gene in familial PAH suggests that additional genetic or environmental factors are involved in clinical manifestation of PAH. Smurf1 ubiquitin ligase, together with inhibitory SMAD 6/7, forms a negative feedback loop for the attenuation of BMP signals by downregulating BMPR and signaling molecules and, in addition, functions in the integration of MAPK/Ras mitogenic pathways. The present study found that Smurf1 was significantly elevated in pulmonary arteries of monocrotaline and hypoxia-induced PAH rats. In the pulmonary artery of hypoxia-exposed mice, elevation of Smurf1 and SMAD7 was correlated with reduced expression of BMPR II protein. Over-expression of Smurf1 in cultured cells induced ubiquitination and degradation of BMPR I and II whereas ligase-inactive Smurf1 reduced ubiquitination and elevated their protein levels, thus serving a dominant-negative function. Smurf1-induced receptor degradation was inhibited by both proteasomal and lysosomal inhibitors. Thus, Smurf1 reduces steady-state levels of BMPRs by ubiquitination and subsequent degradation involving proteasomes and lysosomes. Therefore, these results show that Smurf1 induction could be a key event for triggering downregulation of BMP signaling and causing vascular cell proliferation and remodeling in PAH and that abrogating Smurf1 function could be a strategy for PAH therapeutics.

Hippocampal spine-associated Rap-specific GTPase-activating protein induces enhancement of learning and memory in postnatally hypoxia-exposed mice

Spine-associated Rap-specific GTPase-activating protein (SPAR) is a postsynaptic protein that forms a complex with postsynaptic density (PSD)-95 and N-methyl- d-aspartate receptors (NMDARs), and morphologically regulates dendritic spines. Mild intermittent hypoxia (IH, 16.0% O 2, 4 h/day for 4 weeks) is known to markedly enhance spatial learning and memory in postnatal developing mice. Here, we report that this effect is correlated with persistent increases in SPAR expression as well as long-term potentiation (LTP) in the hippocampus of IH-exposed mice. Furthermore, an infusion of SPAR antisense oligonucleotides into the dorsal hippocampus disrupted elevation of SPAR expression, preventing enhanced hippocampal LTP in IH-exposed developing mice and also reducing LTP in normoxic mice, without altering basal synaptic transmission. In SPAR antisense-treated mice, acquisition of the Morris water maze spatial learning task was impaired, as was memory retention in probe trails following training. This study provides the first evidence that SPAR is functionally required for synaptic plasticity and contributes to the IH-induced enhancement of spatial learning and memory in postnatal developing mice.

Analysis of the effects of exposure to acute hypoxia on oxidative lesions and tumour progression in a transgenic mouse breast cancer model

BackgroundTumour hypoxia is known to be a poor prognostic indicator, predictive of increased risk of metastatic disease and reduced survival. Genomic instability has been proposed as one of the potential mechanisms for hypoxic tumour progression. Both of these features are commonly found in many cancer types, but their relationship and association with tumour progression has not been examined in the same model.MethodsTo address this issue, we determined the effects of 6 week in vivo acute hypoxic exposure on the levels of mutagenic lipid peroxidation product, malondialdehyde, and 8-oxo-7,8-dihydro-2'-deoxyguanosine DNA (8-oxo-dG) lesions in the transgenic polyomavirus middle T (PyMT) breast cancer mouse model.ResultsWe observed significantly increased plasma lipid peroxidation and 8-oxo-dG lesion levels in the hypoxia-exposed mice. Consumption of malondialdehyde also induced a significant increase in the PyMT tumour DNA lesion levels, however, these increases did not translate into enhanced tumour progression. We further showed that the in vivo exposure to acute hypoxia induced accumulation of F4/80 positive tumour-associated macrophages (TAMs), demonstrating a relationship between hypoxia and macrophages in an experimental model.ConclusionThese data suggest that although exposure to acute hypoxia causes an increase in 8-oxo-dG lesions and TAMs in the PyMT tumours, these increases do not translate into significant changes in tumour progression at the primary or metastatic levels in this strong viral oncogene-driven breast cancer model.

Synthesis and pharmacology of pyrid-3-yl sulfonylureas and thioureas as astrocytic Na + 2C1 − K + cotransporter inhibitors

The pharmacology of lipophilic 4-arylamino- and 4-cycloalkylaminopyrid-3-ylsulfonyl(thio)ureas and their synthesis designed from torasemide are described. These compounds could lead to a method of inhibiting the astrocytic Na + 2C1 − K + cotransporter and thus treating cerebral edema. Their lipophilicity and ionization constants were determined. Seven lipophilic compounds ( 17, 18, 22, 23, 24, 32 and 36) exhibited a high inhibitory potency (IC 50 < 10 μM) and had lost the diuretic properties of torasemide. One of them, N-{[4-(cycloheptylamino)pyrid-3-yl[sulfonyl}- N′-cycloheptylurea 17 (0.1-1 mg/kg) was found to increase the gasp delay in hypoxia-exposed mice. In preliminary experiments, 17 (1–2.5 mg/kg) strongly reduced the morbidity and mortality rate of gerbils after permanent left carotid artery ligation. In vitro experiments confirmed its antiedematous activity.

Lymphoidal involution and delayed homograft rejection in hypoxia-exposed mice.

Investigation of the relationship between histologic and cytochemical response patterns of the thymus, spleen, and lymph nodes of mice exposed to moderate hypoxia (380 mm Hg), and study, by histologic analysis, of the effect of hypoxia exposure on the skin homograft reaction used as an index of immunologic potential. The results obtained include the finding that functional changes in lymphatic organs occur during early weeks of hypoxia acclimation and that these changes probably reduce the ability of an animal to react to an immunological challenge.