Meli, A.; (2009) Answers in a flash; optical analysis of exocytosis in human cultured endothelial cells. Doctoral thesis , UCL (University College London).

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Abstract

Endothelial cells line all of our blood vessels. They monitor and respond to signals generated during injury, infection and disease by releasing a wide range of molecules that regulate blood flow, coagulation, inflammatory responses and vessel growth. Protein mediators are released by exocytosis of intracellular organelles, and a major trigger for this type of secretion is an increase in intracellular free calcium ion concentration ([Ca2+]i). Mitochondria are thought to influence Ca2+ homeostasis through local Ca2+ buffering. Due to a lack of sensitive and time-resolved assays for endothelial exocytosis little is known about the precise relationship between Ca2+-signalling and exocytosis, and the influence of Ca2+ buffering by mitochondria. Using fluorescence and biochemical techniques I have investigated the relationship between secretagogue-evoked Ca2+-signalling and the influence of mitochondrial function on the exocytosis of two distinct organelle populations in cultures of Human Umbilical Vein Endothelial Cells (HUVEC); 1) the Weibel-Palade body (WPB) the main storage organelle for pro-coaguland and inflammatory mediators, and 2) the non-WPB, a non-stored and morphologically distinct organelle that can contain a range of inflammatory and anti-coagulant molecules. These two distinct organelle populations were labeled for fluorescence microscopy by targeted expression of chimeras of green (EGFP) or red (mRFP) fluorescent proteins in living HUVEC. Exocytosis was evoked by both physiological and pharmacological secretogogues that increase [Ca2+]i. The times of exocytosis of individual organelles were monitored by flashes of light from granule EGFP, produced by pH changes within the organelle upon fusion. In the same experiments, [Ca2+]i and intra-mitochondrial Ca2+ concentration ([Ca2+]m) were monitored using fluorescent Ca2+-indicators. The data obtained has defined more precisely the relationship between agonist-evoked changes in [Ca2+]i and secretory vesicle exocytosis in HUVEC. These studies will contribute to a better understanding of the processes that regulate secretion of biomolecules from the endothelium.

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