Discoidin Domain receptor 2 signaling in liver sinusoidal endothelial cells during fibrosis – DDR2LSEC

The liver is a vital organ and a site of extraordinary exchange between blood from digestion and hepatocytes. These exchanges are made possible by a unique architecture of the liver capillaries, also called sinusoids, and by specific characteristic of liver sinusoidal endothelial cells (LSECs). LSECs form a morphologically and functionally distinct endothelium with a discontinuous basement membrane and displays fenestrae organized in sieve plate. Fenestrae are small structures (100-200 nm in diameter) only visible using electron microscopy. Liver fibrosis results from chronic damage to the liver and is defined by accumulation of excess extracellular matrix (ECM) components, notably, type I collagen fibrils. Moreover, all forms of fibrosis are characterized by a defenestrated sinusoidal endothelium and formation of a continuous basal membrane corresponding to a phenomenon called capillarization. Recent data obtained from animal models indicate that defenestration may contribute to fibrogenesis. These data highlight the LSEC involvement in control of liver homeostasis.
ECM components regulate actin cytoskeleton organization, which orchestrates a plethora of cellular events as adhesion, differentiation, division and migration. Moreover it was demonstrated that actin filaments surround and regulate LSEC fenestrae. For these reasons, we investigated actin cytoskeleton reorganization in LSECs in response to modifications of its microenvironment. We showed that type I collagen only in its physiological organization in fibrils promote formation on a new class of invadosomes, that we called linear invadosomes. Invadosomes are F-actin structures able to degrade the ECM. Recently, we established that discoidin domain receptor 2 (DDR2), which is a type I collagen receptor is involved in linear invadosome formation in LSECs. These data demonstrate that DDR2 signaling is implicated in actin cytoskeleton reorganization in LSECs. Based on these data, our project aims at investigating DDR2 involvement not only in linear invadosome formation and function, but also in fenestrae organization. Consequently, the project is organized around two objectives.
The first objective aims at investigating DDR2 signaling pathways and partners involved in linear invadosome formation upon contact with type I collagen fibrils. We will develop both a global and a candidate approach. We also plan to analyze the presence of linear invadosomes in vivo, in fibrotic liver sections.
The second objective of our project aims at studying the DDR2 involvement in LSEC fenestrae formation. Recently, we used high-resolution microscopy approach to visualize fenestrae in fluorescence microscopy in fixed LSECs. Thus, the first challenge is to develop this technique to monitor the dynamic and the formation of LSEC fenestrae in living cells. Using a knockdown approach, we will then address the direct involvement of DDR2 in LSEC fenestrae formation. Finally, faced to the crucial absence of molecular markers for these structures, using mass spectrometry, we will set-up a novel approach to identify their composition. This may help to decipher the mechanisms involved in their formation, control and stabilization.
To carry out this ambitious project, we will combine the expertise of specialists in fluorescent, super-resolution and electron microscopy as well as in mass spectrometry.
In the end, this multidisciplinary project should allow a better understanding of the role of LSECs in liver fibrosis.