Lipidome of Activated Hepatic Stellate Cells Figure 1. HSC activation results in redistribution of LDs and is accompanied by a decrease in size. A. Freshly isolated HSCs were cultured and fixed after 2 h (day 0), and 4, 7 or 14 days. Morphology and neutral lipid content was analyzed by differential interference contrast microscopy (DIC) and fluorescence microscopy after Bodipy staining of LDs. Arrows indicate LD redistribution. B. Total number of LDs in HSCs at day 0, 4, 7 and 14 and the area per LD was quantified by Image J software. The results represent the means 6 SEM of 10 representative cells. doi:10.1371/journal.pone.0034945.g001 process. In other words can HSC activation be altered when formation or breakdown of lipid droplets is disturbed? In order to answer this question first a more fundamental knowledge on the molecular mechanism of lipid droplet homeostasis in HSC is required as this is largely lacking at the moment. To acquire more insight in the mechanism of LD loss, and its role in HSC activation, we investigated the LD degradation process and lipidomic alterations in these cells with a combined approach of Raman confocal microspectroscopy and high performance liquid chromatography (HPLC)-coupled mass spectrometry (MS). Ra- man microspectroscopy - a spectroscopic technique based on inelastic scattering of monochromatic light - does not require labeling of the molecules of interest and enables direct specific chemical imaging of biomolecules such as DNA/RNA, proteins, and lipids in intact cells and tissues [13,14]. More importantly, it provides detailed information about the molecular composition of the subcellular volume being probed [15]. Together with a newly developed MS technique enabling analysis of all neutral lipid classes in a single run, we could follow LDs morphologically and biochemically during the HSC activation process in vitro. Isolation of HSCs and subsequent culturing on a plastic surface induces phenotypical alterations resembling the in vivo HSC activation process and is therefore commonly used as a model to study HSC activation. Here, we report that HSCs display a homogeneous LD population with respect to their chemical content. Upon activation LDs drastically change in size, intracellular location and lipidome. Especially a preferential decrease in retinyl esters was observed in the early stages of activation that was accompanied by a specific increase in triglycerides containing polyunsaturated fatty acids. This shows that lipid droplet degradation in activated hepatic stellate cells is a highly dynamic and regulated process. Results Redistribution of lipid droplets during HSC activation To investigate the dynamics of the LD degradation process, freshly isolated HSCs were cultured and fixed at several time points, where after LD morphology and distribution was analysed. In its quiescent form, the HSC is relatively small and star-shaped (Fig. 1A-day 0). During the activation process, the HSC forms cellular extensions (Fig. 1A-day7 arrow), until its final transfor- mation into a myofibroblastic cell (Fig. 1A-day 14). a-smooth muscle actin staining, commonly used as a HSC activation marker, confirmed the activated state of HSCs at day 7 and 14 (not shown). In the quiescent HSC numerous large LDs were observed perinuclearly (Fig. 1A-day 0). In HSCs around day 4 after plating an increased number of smaller LDs was visible, PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e34945