Monitoring Endothelial Cell Activation In Vivo during Thrombus Formation.

The endothelium serves as a metabolically active interface between the blood and underlying tissues and offers response to changes in its microenvironment. The endothelium is rapidly activated in response to stimuli associated with fluctuations in shear stress, physical trauma, oxidative stress, and thrombotic and inflammatory mediators. To study endothelial cell activation we have monitored calcium mobilization in vitro in cultured endothelial cells and also in situ in the living mouse, following vessel wall injury. The addition of exogenous ADP (10 μM) or thrombin (1 U/mL) to Human Umbilical Vein Endothelial Cells (HUVECs) loaded with the calcium sensitive dye, Fluo-4 AM, is followed by rapid elevation of intracellular calcium, with a sustained peak observed within 10 sec. To further investigate the ability of the endothelium to activate in response to mediators potentially localized in the microenvironment of the endothelium during thrombus formation, we investigated the ability of IL-6 (0.1 ng/mL), IL-8 (80 ng/mL), MCP-1 (15 ng/mL), NAP-2 (10 ng/mL) and SDF-α (60 ng/mL) to mobilize calcium in HUVECs. These potential agonists were selected based on their primary roles in inflammation and thrombosis, and proposed roles in atherosclerosis and tumor angiogenesis. SDF-α and NAP-2 rapidly mobilized calcium in HUVECs, with similar maximum responses, but NAP-2 initiated a more prolonged (4-fold longer) rise in intracellular calcium. IL-6, IL-8, and MCP-1 also mobilized calcium, but the rise in intracellular calcium was almost 4-fold lower than that observed with SDF-α and NAP-2. Comparable calcium mobilization occurs in HUVECs subjected to a single pulse of a nitrogen dye-tuned laser. In addition, targeting of single cells within a confluent culture of endothelial cells initiated calcium elevation in the targeted cell and was followed by a wave of calcium elevation in surrounding cells. To determine whether this endothelial cell activation, and more specifically the calcium elevation, occurs in vivo, we conducted experiments using the laser-induced model of thrombus formation to look for calcium elevation in the arteriolar endothelium in live mice. Calcium elevation was monitored by Fluo-4AM introduced intravenously into the mouse circulation via the jugular vein. Fluo-4AM is non-specific in its uptake among cell types, and endothelial cell uptake of the dye in the cremaster muscle microcirculation was observed. Upon laser-induced injury, calcium elevation in the endothelium could be monitored by intravital microscopy. The observed calcium elevation was rapid (within 30 s) and preceded detection of platelets in the developing thrombus. These results add to the evidence of a dynamic endothelium and demonstrate that the endothelium activates rapidly prior to thrombus formation in the laser induced thrombosis model.