Pulmonary hypertension after prolonged hypoxic exposure in mice with a congenital deficiency of Cyp2j.

Cytochrome P450 epoxygenase-derived epoxyeicosatrienoic acids contribute to the regulation of pulmonary vascular tone and hypoxic pulmonary vasoconstriction. We investigated whether the attenuated acute vasoconstrictor response to hypoxic exposure of Cyp2j(-/-) mice would protect these mice against the pulmonary vascular remodeling and hypertension associated with prolonged exposure to hypoxia. Cyp2j(-/-) and Cyp2j(+/+) male and female mice continuously breathed an inspired oxygen fraction of 0.21 (normoxia) or 0.10 (hypoxia) in a normobaric chamber for 6 weeks. We assessed hemoglobin (Hb) concentrations, right ventricular (RV) systolic pressure (RVSP), and transthoracic echocardiographic parameters (pulmonary acceleration time [PAT] and RV wall thickness). Pulmonary Cyp2c29, Cyp2c38, and sEH mRNA levels were measured in Cyp2j(-/-) and Cyp2j(+/+) male mice. At baseline, Cyp2j(-/-) and Cyp2j(+/+) mice had similar Hb levels and RVSP while breathing air. After 6 weeks of hypoxia, circulating Hb concentrations increased but did not differ between Cyp2j(-/-) and Cyp2j(+/+) mice. Chronic hypoxia increased RVSP in Cyp2j(-/-) and Cyp2j(+/+) mice of either gender. Exposure to chronic hypoxia decreased PAT and increased RV wall thickness in both genotypes and genders to a similar extent. Prolonged exposure to hypoxia produced similar levels of RV hypertrophy in both genotypes of either gender. Pulmonary Cyp2c29, Cyp2c38, and sEH mRNA levels did not differ between Cyp2j(-/-) and Cyp2j(+/+) male mice after breathing at normoxia or hypoxia for 6 weeks. These results suggest that murine Cyp2j deficiency does not attenuate the development of murine pulmonary vascular remodeling and hypertension associated with prolonged exposure to hypoxia in mice of both genders.

Multiple wall in-folds sub-divide single segments during capillary regression in hyperoxic acute lung injury.

The present study provides further insight into the structural processes that remodel pulmonary capillaries in the injured adult lung. Early in hyperoxia acute lung injury (HALI), many sub-dividing segments are present throughout the capillary network before segment occlusion and loss predominate and capillary density decreases later in the period. A second segment sub-division triggered in regenerating capillaries after air breathing (post-HALI) demonstrates a similar mechanism of organization at a time of contrasting change in the capillary density. As we have previously reported, the process of segment sub-division includes in-folding of the endothelial-epithelial surface (alveolar-capillary membrane) to form inter-luminal structures (ILSs) and loops, with loop separation increasing segment number. Unexpectedly, the findings support remodeling of the capillary density by wall in-folding in acute lung injury, demonstrating a similar mechanism in capillary regression as well as in regeneration in the adult lung.

Mechanisms of growth of a pulmonary capillary network in adult lung.

The present study provides new insight into structural processes remodeling pulmonary capillaries in adult lung. The data highlight mechanisms underlying the expansion and increased density of capillary segments on return to air breathing (FiO2 0.21) after injury in high oxygen (FiO2 0.75). As segments expand and increase in number, endothelial cells extend their processes to bridge the lumen and support the walls of developing interluminal structures (ILSs); endothelial-epithelial surfaces infold as a single unit (sheet) into the lumen, increasing the length of each surface and subdividing segments by loop formation and by the formation of ILSs; segments further increase in number as lumen subdivision proceeds by intussusceptive microvascular growth (IMG).

Alveolar oxygen tension and angio-architecture of the distal adult lung.

The present study demonstrates the fine structure of pulmonary capillaries first injured and then undergoing growth in response to a change in the ambient alveolar oxygen tension. Breathing a high fraction of inspired oxygen (FiO2 0.75) triggers restriction by endothelial cell injury and effacement leading to segment narrowing and shortening and segment loss as demonstrated by a fall in density. Subsequently, breathing a relatively low fraction (FiO2 0.21) triggers capillary assembly (angiogenesis), which reverses the changes. The data underscore the structural reprogramming (reduction and restoration) of pulmonary capillaries in response to significant shifts in oxygen tension.

A quantitative ultrastructural study of circulating (monocytic) cells interacting with endothelial cells in high oxygen-injured and spontaneously re-forming (FVB) mouse lung capillaries.

The present study demonstrates the fine structure of blood-borne (monocytic) circulating cells (CCs), and their interaction with endothelial cells, in a mouse model of lung capillary injury and repair. Quantitative analysis highlights the diversity of CC profiles entering the lung, where they form contact and adhesion/fusion sites to endothelial plasmalemmal membranes, and to complexes of endothelial/basement membrane remnants, as capillary networks reorganize over time. Temporal patterns of CC influx and efflux in the lung, changing CC phenotypes, and the range of CC interactions with endothelium, underscore the potential for a complex angiogenic/immunogenic response, as capillary networks stabilize and undergo expansion and growth.

A protocol for a lung neovascularization model in rodents.

By providing insight into the cellular events of vascular injury and repair, experimental model systems seek to promote timely therapeutic strategies for human disease. The goal of many current studies of neovascularization is to identify cells critical to the process and their role in vascular channel assembly. We propose here a protocol to analyze, in an in vivo rodent model, vessel and capillary remodeling (reorganization and growth) in the injured lung. Sequential analyses of stages in the assembly of vascular structures, and of relevant cell types, provide further opportunities to study the molecular and cellular determinants of lung neovascularization.

A protocol for phenotypic detection and characterization of vascular cells of different origins in a lung neovascularization model in rodents.

The goal of many current studies of neovascularization is to define the phenotype of vascular cell populations of different origins and to determine how such cells promote assembly of vascular channel. Here, we describe a protocol to immunophenotype vascular cells by high-resolution imaging and by fluorescence-activated flow cytometry in an in vivo rodent model of pulmonary microvascular remodeling. Analysis of cells by this combined approach will characterize their phenotype, quantify their number and identify their role in the assembly of vascular channels.

5-Lipoxygenase deficiency prevents respiratory failure during ventilator-induced lung injury.

RATIONALE: Mechanical ventilation with high VT (HVT) progressively leads to lung injury and decreased efficiency of gas exchange. Hypoxic pulmonary vasoconstriction (HPV) directs blood flow to well-ventilated lung regions, preserving systemic oxygenation during pulmonary injury. Recent experimental studies have revealed an important role for leukotriene (LT) biosynthesis by 5-lipoxygenase (5LO) in the impairment of HPV by endotoxin. OBJECTIVES: To investigate whether or not impairment of HPV contributes to the hypoxemia associated with HVT and to evaluate the role of LTs in ventilator-induced lung injury. METHODS: We studied wild-type and 5LO-deficient mice ventilated for up to 10 hours with low VT (LVT) or HVT. RESULTS: In wild-type mice, HVT, but not LVT, increased pulmonary vascular permeability and edema formation, impaired systemic oxygenation, and reduced survival. HPV, as reflected by the increase in left pulmonary vascular resistance induced by left mainstem bronchus occlusion, was markedly impaired in animals ventilated with HVT. HVT ventilation increased bronchoalveolar lavage levels of LTs and neutrophils. In 5LO-deficient mice, the HVT-induced increase of pulmonary vascular permeability and worsening of respiratory mechanics were markedly attenuated, systemic oxygenation was preserved, and survival increased. Moreover, in 5LO-deficient mice, HVT ventilation did not impair the ability of left mainstem bronchus occlusion to increase left pulmonary vascular resistance. Administration of MK886, a 5LO-activity inhibitor, or MK571, a selective cysteinyl-LT(1) receptor antagonist, largely prevented ventilator-induced lung injury. CONCLUSIONS: These results indicate that LTs play a central role in the lung injury and impaired oxygenation induced by HVT ventilation.

BMPR-II heterozygous mice have mild pulmonary hypertension and an impaired pulmonary vascular remodeling response to prolonged hypoxia.

Heterozygous mutations of the bone morphogenetic protein type II receptor (BMPR-II) gene have been identified in patients with primary pulmonary hypertension. The mechanisms by which these mutations contribute to the pathogenesis of primary pulmonary hypertension are not fully elucidated. To assess the impact of a heterozygous mutation of the BMPR-II gene on the pulmonary vasculature, we studied mice carrying a mutant BMPR-II allele lacking exons 4 and 5 (BMPR-II(+/-) mice). BMPR-II(+/-) mice had increased mean pulmonary arterial pressure and pulmonary vascular resistance compared with their wild-type littermates. Histological analyses revealed that the wall thickness of muscularized pulmonary arteries (<100 mum in diameter) and the number of alveolar-capillary units were greater in BMPR-II(+/-) than in wild-type mice. Breathing 11% oxygen for 3 wk increased mean pulmonary arterial pressure, pulmonary vascular resistance, and hemoglobin concentration to similar levels in BMPR-II(+/-) and wild-type mice, but the degree of muscularization of small pulmonary arteries and formation of alveolar-capillary units were reduced in BMPR-II(+/-) mice. Our results suggest that, in mice, mutation of one copy of the BMPR-II gene causes pulmonary hypertension but impairs the ability of the pulmonary vasculature to remodel in response to prolonged hypoxic breathing.

Myeloperoxidase-associated tyrosine nitration after intratracheal administration of lipopolysaccharide in rats.

BACKGROUND: Previous studies have shown that lipopolysaccharide-induced inflammation in the lung results in tyrosine nitration. The objective of this study was to evaluate the contribution of myeloperoxidase and peroxynitrite pathway to the tyrosine nitration in lipopolysaccharide-administered lungs of rats that were otherwise untreated or leukocyte-depleted by cyclophosphamide or received inhaled nitric oxide (NO). METHODS: The authors analyzed the immunoreactivity of inducible nitric oxide synthase (iNOS), nitrotyrosine (a product of the myeloperoxidase or peroxynitrite pathway), and chlorotyrosine (a byproduct of the myeloperoxidase pathway) by use of specific antibodies. The number of neutrophils in bronchoalveolar lavage fluid (BALF) and levels of myeloperoxidase activity in lung homogenates were also measured. RESULTS: Lipopolysaccharide enhanced the immunoreactivity of iNOS, nitrotyrosine, and chlorotyrosine in alveolar wall cells, alveolar macrophages, and neutrophils. Leukocyte depletion by cyclophosphamide and inhibition of leukocyte accumulation in the lungs by NO inhalation did not eliminate the increase in iNOS immunoreactivity in alveolar macrophages after lipopolysaccharide treatment, but nitrotyrosine and chlorotyrosine were not produced in these cells. Tyrosine nitration in response to lipopolysaccharide was associated with increases in neutrophil count in BALF and in myeloperoxidase activity in lung homogenates, whereas NO inhalation suppressed the neutrophil count in BALF and reduced tyrosine nitration and chlorination. CONCLUSIONS: These findings suggest that myeloperoxidase pathway has a role in tyrosine nitration in the lungs of lipopolysaccharide-treated rats, and that NO inhalation during early phase of inflammation does not increase but rather decreases tyrosine nitration and chlorination, possibly by reducing neutrophil sequestration.

Cytosolic phospholipase A(2) in hypoxic pulmonary vasoconstriction.

Cytosolic phospholipase A(2) (cPLA(2)) releases arachidonic acid (AA) from phospholipids in cell membranes. To assess the role of cPLA(2) in hypoxic pulmonary vasoconstriction (HPV), we measured the increase in left lung pulmonary vascular resistance (LPVR) before and during hypoxia produced by left main stem bronchus occlusion (LMBO) in mice with and without a targeted deletion of the PLA2g4a gene that encodes cPLA(2alpha). LMBO increased LPVR in cPLA(2alpha)(+/+) mice but not in cPLA(2alpha)(-/-) mice. cPLA(2alpha)(+/+) mice were better able to maintain systemic oxygenation during LMBO than were cPLA(2alpha)(-/-) mice. Administration of a cPLA(2) inhibitor, arachidonyl trifluoromethyl ketone, blocked the LMBO-induced increase in LPVR in wild-type mice, while exogenous AA restored HPV in cPLA(2alpha)(-/-) mice. Intravenous angiotensin II infusion increased PVR similarly in cPLA(2alpha)(+/+) and cPLA(2alpha)(-/-) mice. Inhibitors of cyclooxygenase or nitric oxide synthase restored HPV in cPLA(2alpha)(-/-) mice. Breathing 10% oxygen for 3 weeks produced less right ventricular hypertrophy in cPLA(2alpha)(-/-) than in cPLA(2alpha)(+/+) mice, but restored HPV in cPLA(2alpha)(-/-) mice despite the continued absence of cPLA(2) activity. These results indicate that cPLA(2) contributes to the murine pulmonary vasoconstrictor response to hypoxia. Augmenting pulmonary vascular tone restores HPV in the absence of cPLA(2) activity.