Lung capillary endothelial cells (ECs) are a critical target of oxygen toxicity and play a central role in the pathogenesis of hyperoxic lung injury. To determine mechanisms and time course of EC activation in normobaric hyperoxia, we measured endothelial concentration of reactive oxygen species (ROS) and cytosolic calcium ([Ca(2+)](i)) by in situ imaging of 2',7'-dichlorofluorescein (DCF) and fura 2 fluorescence, respectively, and translocation of the small GTPase Rac1 by immunofluorescence in isolated perfused rat lungs. Endothelial DCF fluorescence and [Ca(2+)](i) increased continuously yet reversibly during a 90-min interval of hyperoxic ventilation with 70% O(2), demonstrating progressive ROS generation and second messenger signaling. ROS formation increased exponentially with higher O(2) concentrations. ROS and [Ca(2+)](i) responses were blocked by the mitochondrial complex I inhibitor rotenone, whereas inhibitors of NAD(P)H oxidase and the intracellular Ca(2+) chelator BAPTA predominantly attenuated the late phase of the hyperoxia-induced DCF fluorescence increase after > 30 min. Rac1 translocation in lung capillary ECs was barely detectable at normoxia but was prominent after 60 min of hyperoxia and could be blocked by rotenone and BAPTA. We conclude that hyperoxia induces ROS formation in lung capillary ECs, which initially originates from the mitochondrial electron transport chain but subsequently involves activation of NAD(P)H oxidase by endothelial [Ca(2+)](i) signaling and Rac1 activation. Our findings demonstrate rapid activation of ECs by hyperoxia in situ and identify mechanisms that may be relevant in the initiation of hyperoxic lung injury.

Abstract-Although the formation of hydrostatic lung edema is generally attributed to imbalanced Starling forces, recent data show that lung endothelial cells respond to increased vascular pressure and may thus regulate vascular permeability and edema formation. In combining real-time optical imaging of the endothelial Ca 2ϩ concentration ([Ca 2ϩ ] i ) and NO production with filtration coefficient (K f ) measurements in the isolated perfused lung, we identified a series of endothelial responses that constitute a negative-feedback loop to protect the microvascular barrier. Elevation of lung microvascular pressure was shown to increase endothelial [Ca 2ϩ ] i via activation of transient receptor potential vanilloid 4 (TRPV4) channels. The endothelial [Ca 2ϩ ] i transient increased K f via activation of myosin light-chain kinase and simultaneously stimulated NO synthesis. In TRPV4 deficient mice, pressure-induced increases in endothelial [Ca 2ϩ ] i , NO synthesis, and lung wet/dry weight ratio were largely blocked. Endothelial NO formation limited the permeability increase by a cGMP-dependent attenuation of the pressure-induced [Ca 2ϩ ] i response. Inactivation of TRPV4 channels by cGMP was confirmed by whole-cell patch-clamp of pulmonary microvascular endothelial cells and intravital imaging of endothelial [Ca 2ϩ ] i . Hence, pressure-induced endothelial Ca 2ϩ influx via TRPV4 channels increases lung vascular permeability yet concomitantly activates an NO-mediated negative-feedback loop that protects the vascular barrier by a cGMP-dependent attenuation of the endothelial [Ca 2ϩ ] i response. The identification of this novel regulatory pathway gives rise to new treatment strategies, as demonstrated in vivo in rats with acute myocardial infarction in which inhibition of cGMP degradation by the phosphodiesterase 5 inhibitor sildenafil reduced hydrostatic lung edema. Key Words: pulmonary edema Ⅲ vascular permeability Ⅲ vascular endothelium Ⅲ phosphodiesterase type 5 inhibitor Ⅲ nitric oxide T he pathogenesis of hydrostatic lung edema has been attributed predominantly to an imbalance in Starling forces, ie, fluid extravasation attributable to an increased hydrostatic or reduced oncotic pressure gradient across the microvascular barrier. This classic view has been challenged by the findings of Parker and Ivey in isolated perfused rat lungs, which demonstrated an increase in lung filtration coefficient (K f ) following elevation of left atrial pressure (P LA ). 1 This increase was attenuated by the ␤-adrenergic agonist isoproterenol, indicating that the K f increase was not only caused by an enlarged vascular surface area but also resulted from an increase in vascular permeability that could be counteracted via the cAMP signaling pathway. The latter finding suggests that active endothelial responses may contribute critically to the formation of hydrostatic lung edema.By use of real-time fluorescence imaging techniques, we recently identified such endothelial responses to an acute elevation in hydrostatic pres...

Hypoxic pulmonary vasoconstriction (HPV) is a physiological mechanism by which pulmonary arteries constrict in hypoxic lung areas in order to redirect blood flow to areas with greater oxygen supply. Both oxygen sensing and the contractile response are thought to be intrinsic to pulmonary arterial smooth muscle cells. Here we speculated that the ideal site for oxygen sensing might instead be at the alveolocapillary level, with subsequent retrograde propagation to upstream arterioles via connexin 40 (Cx40) endothelial gap junctions. HPV was largely attenuated by Cx40-specific and nonspecific gap junction uncouplers in the lungs of wildtype mice and in lungs from mice lacking Cx40 (Cx40 -/-). In vivo, hypoxemia was more severe in Cx40 -/-mice than in wild-type mice. Real-time fluorescence imaging revealed that hypoxia caused endothelial membrane depolarization in alveolar capillaries that propagated to upstream arterioles in wild-type, but not Cx40 -/-, mice. Transformation of endothelial depolarization into vasoconstriction involved endothelial voltage-dependent α 1G subtype Ca 2+ channels, cytosolic phospholipase A 2 , and epoxyeicosatrienoic acids. Based on these data, we propose that HPV originates at the alveolocapillary level, from which the hypoxic signal is propagated as endothelial membrane depolarization to upstream arterioles in a Cx40-dependent manner. IntroductionHypoxic pulmonary vasoconstriction (HPV) is a fundamental physiological mechanism by which the lung optimizes ventilation/ perfusion (V/Q) matching, redirecting blood flow from poorly to better ventilated areas (1). Yet in cases of global hypoxia, HPV may unfavorably increase total pulmonary vascular resistance and right ventricular afterload, thus contributing to the clinical pathology of pulmonary hypertension and cor pulmonale in chronic hypoxic lung diseases or to pulmonary edema at high altitude (1, 2). While the relevance of HPV has been recognized for over 60 years, the underlying oxygen sensing and signal transduction processes remain a topic of intense research and controversy. Current concepts of HPV are largely based on the notion that pulmonary arterial smooth muscle cells (PASMCs) constitute both the sensor and the transducer of the hypoxic signal as well as its contractile effector (1), while the role of the vascular endothelium is at best considered that of a modulating bystander.From a conceptual standpoint, the ideal site for an oxygen sensor in HPV is within the actual area of pulmonary gas exchange,

Intravital microscopy (IVM) is considered as the gold standard for in vivo investigations of dynamic microvascular regulation. The availability of transgenic and knockout animals has propelled the development of murine IVM models for various organs, but technical approaches to the pulmonary microcirculation are still scarce. In anesthetized and ventilated BALB/c mice, we established a microscopic access to the surface of the right upper lung lobe by surgical excision of a window of 7- to 10-mm diameter from the right thoracic wall. The window was covered by a transparent polyvinylidene membrane and sealed with alpha-cyanoacrylate. Removal of intrathoracic air via a trans-diaphragmal intrapleural catheter coupled the lung surface to the window membrane. IVM preparations were hemodynamically stable for at least 120 min, with mean arterial blood pressure above 70 mmHg, and mean arterial Po(2) and arterial Pco(2) in the range of 90-100 Torr and 30-40 Torr, respectively. Imaged lungs did not show any signs of acute lung injury or edema. Following infusion of FITC dextran, subpleural pulmonary arterioles and venules of up to 50-microm diameter and alveolar capillary networks could be visualized during successive expiratory plateau phases over a period of at least 2 h. Vasoconstrictive responses to hypoxia (11% O(2)) or infusion of the thromboxane analog U-46619 were prominent in medium-sized arterioles (30- to 50-microm diameter), minor in small arterioles <30 microm, and absent in venules. The presented IVM model may constitute a powerful new tool for investigations of pulmonary microvascular responses in mice.