CALCIUM REGULATION IN LUNG MICROVASCULAR ENDOTHELIUM

Columbia University Health Sciences, New York NY

Investigators

Linked publications & trials

Abstract

DESCRIPTION (provided by applicant): Aims: Our overall aim is to understand the in situ biology of lung endothelial cells (EC) in relation to the initiation of lung inflammation. We reported that mitochondrial H2O2 determines endothelial proinflammatory activation. Here, we will determine modification of the H2O2 effect by nitric oxide (NO). The specific aims are to determine in EC of lung capillaries (i) the extent to which NO modifies reactive oxygen species (ROS) formation, and (ii) NO-ROS interactions determining cell-surface expression of P-selectin and TNFalpha receptor type 1. Procedures: We will use the isolated, blood-perfused lung preparation for mouse and rat. Arteriolar, septal and venular capillaries will be loaded with specific fluorophores for the detection of cytosolic, mitochondrial and endosomal store Ca2+, for ROS and NO production and for detection of expression and shedding of P-selectin and TNFR1. Single EC in these capillaries will be optically imaged in real-time by of wide-angle and confocal microscopy. For proinflammatory activation, the capillaries will be challenged with TNFalpha, high capillary pressure, arachidonate and photolytic uncaging of Ca2+. EC responses will be determined with regard to NO and ROS production, and the expression and shedding of the P-selectin and TNFR1. The hypotheses will also be tested in mice containing genetically defective NAD(P)H oxidase (gp91phox(-/-)), and mice deficient in eNOS, iNOS and TNFR1. The data will be processed by means of image analysis software. Significance: Although NO-ROS interactions are well studied in the pathological context, the extent to which such interactions regulate physiological responses of the lung capillary remains unclear. Our research will reveal for the first time, interactions between NO and ROS of mitochondrial and NAD(P)H oxidase origins, in the initiation of proinflammatory events in lung capillaries. We will understand the role of these interactions in regulating proinflammatory receptor expression. These considerations will advance the understanding of the inception of lung inflammation.