Patrice M. Becker

Neuropilin-1 Regulates Vascular Endothelial Growth Factor-Mediated Endothelial Permeability

Neuropilin-1 (Npn-1) is a cell surface receptor that binds vascular endothelial growth factor (VEGF), a potent mediator of endothelial permeability, chemotaxis, and proliferation. In vitro, Npn-1 can complex with VEGF receptor-2 (VEGFR2) to enhance VEGFR2-mediated endothelial cell chemotaxis and proliferation. To determine the role of Npn-1/VEGFR2 complexes in VEGF-induced endothelial barrier dysfunction, endothelial cells were stably transfected with Npn1 or VEGFR2 alone (PAE/Npn and PAE/KDR, respectively), or VEGFR2 and Npn-1 (PAE/KDR/Npn-1). Permeability, estimated by measurement of transendothelial electrical resistance (TER), of PAE/Npn and PAE/KDR cell lines was not altered by VEGF165. In contrast, TER of PAE/KDR/Npn-1 cells decreased in dose-dependent fashion following VEGF165 (10 to 200 ng/mL). Activation of VEGFR2, and 2 downstream signaling intermediates (p38 and ERK1/2 MAPK) involved in VEGF-mediated permeability, also increased in PAE/KDR/Npn-1. Consistent with these data, inhibition of Npn-1, but not VEGFR2, attenuated VEGF165-mediated permeability of human pulmonary artery endothelial cells (HPAE), and VEGF121 (which cannot ligate Npn-1) did not alter TER of HPAE. Npn-1 inhibition also attenuated both VEGF165-mediated pulmonary vascular leak and activation of VEGFR2, p38, and ERK1/2 MAPK, in inducible lung-specific VEGF transgenic mice. These data support a critical role for Npn-1 in regulating endothelial barrier dysfunction in response to VEGF and suggest that activation of distinct receptor complexes may determine specificity of cellular response to VEGF.

Pulmonary epithelial neuropilin-1 deletion enhances development of cigarette smoke-induced emphysema.

RATIONALE Cigarette smoke (CS) exposure is an important risk factor for chronic obstructive pulmonary disease; however, not all smokers develop disease, suggesting that other factors influence disease development. OBJECTIVES We sought to determine whether neuropilin-1 (Nrp1), an integral component of receptor complexes mediating alveolar septation and vascular development, was involved in maintenance of normal alveolar structure, and/or altered susceptibility to the effects of CS. METHODS Transgenic mice were generated to achieve inducible lung-specific deletion of epithelial Nrp1. We determined whether conditional Nrp1 deletion altered airspace size, then compared the effects of chronic CS or filtered air exposure on airspace size, inflammation, and the balance between cell death and proliferation in conditionally Nrp1-deficient adult mice and littermate controls. Finally, we evaluated the effects of Nrp1 silencing on cell death after acute exposure of A549 cells to cigarette smoke extract or short chain ceramides. MEASUREMENTS AND MAIN RESULTS Genetic deletion of epithelial Nrp1 in either postnatal or adult lungs resulted in a small increase in airspace size. More notably, both airspace enlargement and apoptosis of type I and type II alveolar epithelial cells were significantly enhanced following chronic CS exposure in conditionally Nrp1-deficient adult mice. Silencing of Nrp1 in A549 cells did not alter cell survival after vehicle treatment but significantly augmented apoptosis after exposure to cigarette smoke extract or ceramide. CONCLUSIONS These data support a role for epithelial Nrp1 in the maintenance of normal alveolar structure and suggest that dysregulation of Nrp1 expression may promote epithelial cell death in response to CS exposure, thereby enhancing emphysema development.

F2-isoprostane generation in isolated ferret lungs after oxidant injury or ventilated ischemia

Pulmonary edema develops when pulmonary blood flow is interrupted, then restored. Because the lung is not always hypoxic when ischemic, mechanisms of pulmonary ischemia-reperfusion injury are likely to differ from systemic organs, where reactive oxygen species generated during reperfusion mediate organ dysfunction. We previously showed that pulmonary vascular permeability of isolated ferret lungs increased prior to reperfusion, if ventilation was maintained while blood flow was impaired. To determine whether reactive oxygen metabolites generated during ischemia mediated ischemic injury, we measured tissue levels of F2-isoprostanes as an index of lipid peroxidation, 30 min after administration of glucose (5 mM)-glucose oxidase (GOX, 0.1 U/ml), or after short (45 min) or long (180 min) ventilated ischemia, in isolated ferret lungs. Osmotic reflection coefficient for albumin (sigma alb), an estimate of vascular protein permeability, was measured in the same lungs. Tissue F2-isoprostanes increased 375% after exposure to glucose-GOX in association with a 42% decrease in sigma alb, and administration of catalase (CAT, 100,000 U) and superoxide dismutase (SOD, 25,000 U) completely attenuated this lipid peroxidation. In contrast, tissue F2-isoprostanes increased only 60% following 45 min of ischemia, then did not increase additionally. sigma alb was not altered by 45 min of ischemia, but decreased 72% following 180 min of ischemia. CAT+SOD did not alter F2-isoprostane formation during ischemia, but partially attenuated vascular injury. These results suggest that tissue levels of F2-isoprostanes reflect lung lipid peroxidation, but that F2-isoprostane generation does not directly increase vascular permeability following ventilated pulmonary ischemia.

Mechanistic basis for the potent anti-angiogenic activity of semaphorin 3F.

Neuropilin-1 (Nrp1), an essential type I transmembrane receptor, binds two secreted ligand families, vascular endothelial growth factor (VEGF) and class III Semaphorin (Sema3). VEGF-A and Sema3F have opposing roles in regulating Nrp1 vascular function in angiogenesis. VEGF-A functions as one of the most potent pro-angiogenic cytokines, while Sema3F is a uniquely potent endogenous angiogenesis inhibitor. Sema3 family members require proteolytic processing by furin to allow competitive binding to Nrp1. We demonstrate that the furin-processed C-terminal domain of Sema3F (C-furSema) potently inhibits VEGF-A-dependent activation of endothelial cells. We find that this potent activity is due to unique heterobivalent engagement of Nrp1 by two distinct sites in the C-terminal domain of Sema3F. One of the sites is the C-terminal arginine, liberated by furin cleavage, and the other is a novel upstream helical motif centered on the intermolecular disulfide. Using a novel chimeric C-furSema, we demonstrate that combining a single C-terminal arginine with the helical motif is necessary and sufficient for potent inhibition of binding of VEGF-A to Nrp1. We further demonstrate that the multiple furin-processed variants of Sema3A, with the altered proximity of the two binding motifs, have dramatically different potencies. This suggests that furin processing not only switches Sema3 to an activated form but also, depending on the site processed, can also tune potency. These data establish the basis for potent competitive binding of Sema3 to Nrp1 and provide a basis for the design of bivalent Nrp inhibitors.

Interleukin-6 mediates pulmonary vascular permeability in a two-hit model of ventilator-associated lung injury

ABSTRACT To test the hypothesis that interleukin-6 (IL-6) contributes to the development of ventilator-associated lung injury (VALI), IL-6–deficient (IL6−/–) and wild-type control (WT) mice received intratracheal hydrochloric acid followed by randomization to mechanical ventilation (MV + IT HCl) or spontaneous ventilation (IT HCl). After 4 hours, injury was assessed by estimation of lung lavage protein concentration and total and differential cell counts, wet/dry lung weight ratio, pulmonary cell death, histologic inflammation score (LIS), and parenchymal myeloperoxidase (MPO) concentration. Vascular endothelial growth factor (VEGF) concentration was measured in lung lavage and homogenate, as IL-6 and stretch both regulate expression of this potent mediator of permeability. MV-induced increases in alveolar barrier dysfunction and lavage VEGF were attenuated in IL6−/− mice as compared with WT controls, whereas tissue VEGF concentration increased. The effects of IL-6 deletion on alveolar permeability and VEGF concentration were inflammation independent, as parenchymal MPO concentration, LIS, and lavage total and differential cell counts did not differ between WT and IL6−/− mice following MV + IT HCl. These data support a role for IL-6 in promoting VALI in this two-hit model. Strategies to interfere with IL-6 expression or signaling may represent important therapeutic targets to limit the injurious effects of MV in inflamed lungs.

Interactive Effects of Mechanical Ventilation and Kidney Health on Lung Function in an In Vivo Mouse Model

We hypothesized that the influence of acute kidney injury (AKI) on the sensitivity of the lung to an injurious process varies with the severity of the injurious process. Thus, we thought that AKI would exacerbate lung injury from low degrees of lung trauma but attenuate lung injury from higher degrees of lung trauma. C57BL/6 mice underwent AKI (30-min kidney ischemia) or sham surgery, followed at 24 h by 4 h of spontaneous breathing (SB), mechanical ventilation with low tidal volume (7 ml/kg, LTV), or mechanical ventilation with high tidal volume (30 ml/kg, HTV). Compared with LTV, median bronchoalveolar lavage (BAL) protein leak was significantly lower with SB and greater with HTV in both sham and AKI mice. Compared with LTV, median Evans blue dye-labeled albumin extravasation in lungs (L-EBD) was also significantly lower with SB and greater with HTV. L-EBD showed a significant interaction between ventilatory mode and kidney health, such that AKI attenuated the L-EBD rise seen in HTV vs. LTV sham mice. An interaction between ventilatory mode and kidney health could also be seen in BAL neutrophil number (PMN). Thus, AKI attenuated the BAL PMN rise seen in HTV vs. LTV sham mice. These data support the presence of a complex interaction between mechanical ventilation and AKI in which the sensitivity of the lung to trauma varies with the magnitude of the trauma and may involve a modification of pulmonary neutrophil activity by AKI.

Semaphorin 3A Contributes to Distal Pulmonary Epithelial Cell Differentiation and Lung Morphogenesis

Rationale Semaphorin 3A (Sema3A) is a neural guidance cue that also mediates cell migration, proliferation and apoptosis, and inhibits branching morphogenesis. Because we have shown that genetic deletion of neuropilin-1, which encodes an obligatory Sema3A co-receptor, influences airspace remodeling in the smoke-exposed adult lung, we sought to determine whether genetic deletion of Sema3A altered distal lung structure. Methods To determine whether loss of Sema3A signaling influenced distal lung morphology, we compared pulmonary histology, distal epithelial cell morphology and maturation, and the balance between lung cell proliferation and death, in lungs from mice with a targeted genetic deletion of Sema3A (Sema3A-/-) and wild-type (Sema3A+/+) littermate controls. Results Genetic deletion of Sema3A resulted in significant perinatal lethality. At E17.5, lungs from Sema3A-/- mice had thickened septae and reduced airspace size. Distal lung epithelial cells had increased intracellular glycogen pools and small multivesicular and lamellar bodies with atypical ultrastructure, as well as reduced expression of type I alveolar epithelial cell markers. Alveolarization was markedly attenuated in lungs from the rare Sema3A-/- mice that survived the immediate perinatal period. Furthermore, Sema3A deletion was linked with enhanced postnatal alveolar septal cell death. Conclusions These data suggest that Sema3A modulates distal pulmonary epithelial cell development and alveolar septation. Defining how Sema3A influences structural plasticity of the developing lung is a critical first step for determining if this pathway can be exploited to develop innovative strategies for repair after acute or chronic lung injury.

Measurements of Free Radicals in Isolated, Ischemic Lungs and Lung Mitochondria

Previous studies in isolated, ventilated lungs have demonstrated by indirect measurements that oxidant generation occurs during pulmonary ischemia before reperfusion. To identify and quantify the types of free radical species generated during ischemia, we used electron paramagnetic resonance (EPR) spectroscopy in the presence and absence of the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). EPR spectra obtained from the vascular effluent of isolated ferret lungs, contained a doublet signal (g = 2.005) indicative of ascorbyl radical. This signal doubled in magnitude after 180 min of ischemia, providing evidence of oxidant formation during ischemia. When DMPO, which reacts with radicals including superoxide anions and hydroxyl radicals, was added to the perfusate, the spectra contained ascorbyl radical signals but no DMPO-adducts. To clarify the relationship between ascorbyl radical and DMPO-adduct formation, additional studies were conducted in the presence and absence of ascorbate with isolated lung mitochondria as the source of free radicals. The results showed that in the presence of ascorbate, oxygen free radicals were not detected by EPR spin trapping with DMPO because of the formation of prominent ascorbyl radical signals. These data suggest that DMPO may be useful for the detection of reactive oxygen species in isolated lungs, provided the ascorbate can be sufficiently depleted. Alternatively, as shown by our results, EPR studies that directly monitor ascorbyl radical formation may be used as a marker of oxidative stress in the lung.

Cocaine effects on digital blood flow and diffusing capacity for carbon monoxide among chronic cocaine users.

PURPOSE To determine the acute effects of intravenous (i.v.) cocaine on primarily digital skin blood flow and diffusion capacity for carbon monoxide (CO), and secondarily on subjective and cardiovascular measures. PATIENTS AND METHODS A double-blind, Latin-square, placebo-controlled, dose-response study was conducted in an inpatient general clinical research center and clinical pharmacology unit of a university teaching hospital. Twelve adult males with histories of illicit drug use including i.v. cocaine received 0, 25, and 50 mg of i.v. cocaine given as 1-minute infusions, on 3 consecutive test days. Digital cutaneous blood flow was determined via laser doppler flowmetry and skin temperature. Diffusing capacity for carbon monoxide (DCO) was measured with standard techniques. Subjective responses were measured by oral report of a numerical ranking of strength of drug effect. Heart rate and blood pressure responses were measured by electronic sphygmomanometer. RESULTS A maximal decrease in skin blood flow occurred at 2 to 3 minutes after infusion, and was not distinguished among drug conditions. Blood flow returned to baseline more rapidly after placebo than after cocaine: 7 minutes (placebo), 35 minutes (25 mg cocaine), 50 minutes (50 mg cocaine). Skin temperature decreased by 1.25 degrees C after placebo and by 2.75 and 3.25 degrees C after 25 and 50 mg of cocaine, respectively. DCO changed by -1.02 (mean) +/- 0.25 (standard deviation), 0.16 +/- 1.22, and 0.21 +/- 1.63 ml/min/mm Hg following placebo, 25, and 50 mg of cocaine, respectively. Typical subjective, chronotropic, and pressor responses to cocaine were demonstrated, and these occurred in close temporal relationship to digital blood flow and skin temperature responses. CONCLUSIONS The digital cutaneous circulation is highly sensitive to vasoconstrictor effects of cocaine. Pulmonary blood volume tends to be preserved after i.v. cocaine. Subjective effects and cardiovascular responses occur in concert with peripheral blood flow changes. The peripheral vasoconstrictor effects have implications for cocaine users with concurrent vasospastic or vasculopathic disorders.