The role of basement membranes in CNS blood vessel integrity
All blood vessels are ensheathed by extracellular matrix layers known as basement membranes that are crucial for vessel development, stability and barrier function (Sorokin et al., 2010). In the brain, the basement membranes of blood vessels are integral components of the neurovascular unit, but are rarely considered in either the functional integrity of vessels or leukocyte extravasation during neuroinflammation. Defects in CNS vascular basement membranes are associated with developmental defects and increased susceptibility to microvessel stroke. Work of partner 3 has shown that two biochemically and functionally distinct basement membranes are associated with CNS vessels. Of all the basement membrane components, molecules of the laminin family have been shown to be the functionally active components, signalling distinct information that control processes such as cell migration, differentiation and survival. Previous work has shown that laminin α4 and α5 containing isoforms in the endothelial cell basement membrane define sites of leukocyte extravasation into the brain (Wu et al., 2009). Loss of laminin α4 results in a marked and selective reduction of T-lymphocyte infiltration into the brain and reduced susceptibility and severity in EAE. Part of this effect is due to signals transduced to the leukocytes which affects their migration, but more recent data from stroke models (Enzmann et al., 2013) suggest that the laminins also signal to the endothelium, impacting on its barrier function. This project will therefore investigate two main questions: How do different immune cells and endothelial cells bind to the different vascular laminin isoforms and what are the intracellular signals transduced to the cells? Can/how do vascular laminins impact on endothelial cell junction tightness? These questions will be examined through the use of in vitro assays to assess the binding and migration of immune cells and endothelial cells on the vascular laminins and the molecular complexes involved, but also through the use of laminin α4 KO or laminin α5 endothelial cell specific KO mice in models of neuroinflammation and stroke.
- Wu C, Ivars F, Andersson P, Hallmann R, Vestweber D, Nilsson P, Robenek H, Tryggvason K, Song J, Korpos E, Loser K, Beissert S, Georges-Labouesse E Sorokin L (2009) Endothelial basement membrane laminin α5 selectively inhibits T lymphocyte extravasation into the brain. Nat Med 15: 519-527.
- Sorokin, L. (2010). The impact of the extracellular matrix on inflammation. Nature Rev Immunol. 10, 712-723.
- Enzmann, G., Mysiorek, C., Y-J., Gorina, R., Cheng, Ghavampour, S., Hannocks, M-J., Prinz, V., Dirnagl, U., Endres, M., Prinz, M., Beschorner, R., Harter, R., Mittelbronn, M., Engelhardt, B. and Sorokin, L. (2013). The neurovascualr unit as a selective barrier to polymorphonuclear granulocyte (PMN) infitration into the brain after ischemic injury. Acta Neuropathologica 125, 395-412
Tasks and methodology
- In vitro adhesion and migration assays to assess immune and endothelial cell behaviour on different vascular laminin isoforms using techniques such as TIRF microscopy and time lapse video microscopy.
- Flow cytometry to define potential laminin receptors (integrin and non-integrin) expressed by the immune and endothelial cells.
- In vitro adhesion assays to define the molecular complexes (eg integrins or non-integrin receptors) involved in binding immune cells or CNS endothelium to laminin isoforms.
- Immunoprecipitation and immunoblotting experiments to assess intracellular pathways activated by immune or endothelial cell interactions with the different vascular laminins.
- High resolution confocal immunofluorescence imaging of brain sections to localize adhesion complexes in situ in both healthy mice and mice subject to neuroinflammatory (experimental autoimmune encephalomyelitis (EAE)) and stroke models (MCAO).
- High resolution fast fluorescence imaging (spinning disc confocal microscopy) of identified complexes in immune cells and endothelial cells plated on vascular laminins.
Université de Caen-Basse Normandie, Evaluation of basement membrane proteins in mouse stroke model, 8 month