Replacement of Retinyl Esters by Polyunsaturated Triacylglycerol Species in Lipid Droplets of Hepatic Stellate Cells during Activation

Activation of hepatic stellate cells has been recognized as one of the first steps in liver injury and repair. During activation, hepatic stellate cells transform into myofibroblasts with concomitant loss of their lipid droplets (LDs) and production of excessive extracellular matrix. Here we aimed to obtain more insight in the dynamics and mechanism of LD loss. We have investigated the LD degradation processes in rat hepatic stellate cells in vitro with a combined approach of confocal Raman microspectroscopy and mass spectrometric analysis of lipids (lipidomics). Upon activation of the hepatic stellate cells, LDs reduce in size, but increase in number during the first 7 days, but the total volume of neutral lipids did not decrease. The LDs also migrate to cellular extensions in the first 7 days, before they disappear. In individual hepatic stellate cells. all LDs have a similar Raman spectrum, suggesting a similar lipid profile. However, Raman studies also showed that the retinyl esters are degraded more rapidly than the triacylglycerols upon activation. Lipidomic analyses confirmed that after 7 days in culture hepatic stellate cells have lost most of their retinyl esters, but not their triacylglycerols and cholesterol esters. Furthermore, we specifically observed a large increase in triacylglycerol-species containing polyunsaturated fatty acids, partly caused by an enhanced incorporation of exogenous arachidonic acid. These results reveal that lipid droplet degradation in activated hepatic stellate cells is a highly dynamic and regulated process. The rapid replacement of retinyl esters by polyunsaturated fatty acids in LDs suggests a role for both lipids or their derivatives like eicosanoids during hepatic stellate cell activation

The Arabidopsis leucine-rich repeat receptor kinase MIK2/LRR-KISS connects cell wall integrity sensing, root growth and response to abiotic and biotic stresses

Plants actively perceive and respond to perturbations in their cell walls which arise during growth, biotic and abiotic stresses. However, few components involved in plant cell wall integrity sensing have been described to date. Using a reverse-genetic approach, we identified the Arabidopsis thaliana leucine-rich repeat receptor kinase MIK2 as an important regulator of cell wall damage responses triggered upon cellulose biosynthesis inhibition. Indeed, loss-of-function mik2 alleles are strongly affected in immune marker gene expression, jasmonic acid production and lignin deposition. MIK2 has both overlapping and distinct functions with THE1, a malectin-like receptor kinase previously proposed as cell wall integrity sensor. In addition, mik2 mutant plants exhibit enhanced leftward root skewing when grown on vertical plates. Notably, natural variation in MIK2 (also named LRR-KISS) has been correlated recently to mild salt stress tolerance, which we could confirm using our insertional alleles. Strikingly, both the increased root skewing and salt stress sensitivity phenotypes observed in the mik2 mutant are dependent on THE1. Finally, we found that MIK2 is required for resistance to the fungal root pathogen Fusarium oxysporum. Together, our data identify MIK2 as a novel component in cell wall integrity sensing and suggest that MIK2 is a nexus linking cell wall integrity sensing to growth and environmental cues

Lipids on the move : lipid metabolism in protein transport and hepatic stellate cell activation

Lipids are essential in life. They are the building blocks of biomembranes, but are also stored in lipid droplets - to serve as an energy depot and maintain a balanced lipid metabolism. Both these aspects are investigated in this thesis, with the dynamic lipid droplet as a shared key organelle. In the first project, the role of phosphatidylcholine (PC), the major phospholipid of biomembranes, in eukaryotic cellular processes was investigated by using MT58 cells. These cells bear a temperature sensitive mutation in CTP:phosphocholline cytidylyltransferase (CT)?, the regulatory enzyme in the PC synthesis pathway. We showed that inhibition of PC synthesis resulted in impaired protein transport from the Golgi complex towards the plasma membrane in MT58 cells. The decreased PC levels in MT58 cells at the non-permissive temperature are accompanied by a decline of the major DAG species in isolated Golgi membranes. A direct link between PC and DAG levels is suggested by the fact that the decrease of PC species is reflected by a decrease of DAG species with a similar fatty acid composition. Furthermore, the decline in DAG in PC-depleted Golgi membranes results in a disturbed localization of the DAG-binding protein PKD. LysoPC treatment restores DAG concentration, PKD localization and protein transport at the Golgi complex. These findings demonstrate a direct interaction between PC synthesis and DAG levels in the Golgi complex. This interaction proved to be crucial for recruitment and activation of factors involved in Golgi-mediated protein transport, underlining the role of lipids in the secretory pathway. In the second project, differences in the complex neutral lipid composition of (murine) hepatic stellate cells (HSC) during activation were identified by Raman confocal microspectroscopy and a newly adapted HPLC-APCI-MS method. Hepatic stellate cells store vitamin A in lipid droplets (LDs). However, cell activation caused by liver inflammation and repair, results in LD loss. Studying the LD degradation process, we demonstrated that upon activation of the HSCs, the LDs reduced in size, but increased in number and migrated to cellular extensions. The lipid composition in activated HSCs differed strikingly from that in quiescent HSCs. After 7 days in culture HSCs had lost most of their retinyl esters, but not their triacylglycerols (TAGs) and cholesterol esters. Furthermore, we could demonstrate a large increase in the formation of TAG species containing polyunsaturated fatty acids in the activated HSCs. As these TAG-PUFAs are at least in part a result from an enhanced uptake and conversion of arachidonic acid, this suggests a role for eicosanoid metabolism in the HSC activation process. It is clear that the functions of lipids and lipid droplets are more diverse than thought. They also seem to play important roles in an increasing number of diseases. With this research project, we hope to contribute to the mapping of these lipid/lipid droplet functions

Security and Robustness issues in Collaborative Runtime Verification

Decentralized monitors can have robustness and security risks. Among robustness risks are attacks on the monitor's infrastructure in or- der to disable parts of its functionality. Among security risks are attacks that try to extract information from the monitor, and thereby possibly leak sensitive information. Formal methods to analyze these issues given a monitor design can help to make better designs and/or identify crit- ical parts in a monitor. In this paper we specify a model for analyzing robustness and security risks for monitors where a network of runtime local monitors forms a collaborative monitor

Raman imaging and lipidomic analysis of lipid droplets in (activated) hepatic stellate cells

In eukaryotic cells the excess of hydrophobic molecules is stored in special organelles named lipid droplets (LDs). These droplets contain triacylglycerides, cholesteryl esters and/or retinyl esters, depending on the function of the cell in which they reside. Retinyl esters, the storage form of vitamin A, are mainly stored in LDs in hepatic stellate cells (HSC), which comprise about 5–10% of total liver cells