Understanding the Mechanotransductional Basis of Intravascular Bubble Injury

Abstract

Intravascular bubbles, commonly introduced during surgery or precipitated from dissolved gases in decompression sickness, can occlude vessels, diminish perfusion, and initiate thrombotic and inflammatory pathways. The specific interactions between bubbles and the endothelium that lead to cell injury and death are poorly understood. We report live single cell time-lapse imaging of human umbilical vein endothelial cells (HUVECs) perturbed by microbubbles produced by injecting air through a pulled glass capillary ground so that its bevel opposes the buoyant force of the bubble. The bubble is moved with a three-stage micromanipulator into contact with a cell containing Fluo-4, a calcium sensitive dye, and imaged using phase-contrast and fluorescence microscopy. A significant transient elevation in intracellular calcium is observed in response to bubble impact. Membrane depolarization (external K+, 130 mM) does not block the calcium response, indicating that voltage-dependent calcium channels are not involved. Extracellular calcium and an intact actin cytoskeleton are required for the elevation of intracellular calcium upon bubble impact, which is reduced with the addition of gadolinium, a general inhibitor of mechanosensitive channels. These results suggest that a combination of the mechanical deformation and the air-water interface of the bubble activate a plasmalemma ion channel triggering further release of internal calcium stores. The calcium response is ameliorated with addition of surfactant, likely competing for occupancy of the air-liquid bubble interface. Supported by ONR N00014-08-1-0436 and NIH R01 HL67986.