Werner Risau also had a special interest in understanding the development, differentiation and maintenance of the blood-brain barrier (BBB). The BBB is formed on the level of endothelial cells in the central nervous system (CNS). The vascular system of the CNS is derived from endothelial cells which have invaded the early embryonic neuroectoderm from the perineural vascular plexus. Werner Risau ´s focus was to define the key regulators in brain angiogenesis. As none of the molecules involved in brain angiogenesis is uniquely expressed in the CNS, it was clear to him that additional factors must be responsible for the induction of the BBB specific endothelial characteristics such as complex tight junctions, low number of vesicles, and the expression of specialized transport systems. Until today, these molecules are unknown.

Werner Risau and co-workers characterized a cell surface protein named HT7 as an integral membrane glycoprotein of the Ig-superfamily that is expressed specifically on BBB endothelium. Taking advantage of an in vivo model of BBB induction based on the expression of the HT7 cell surface protein, it could be demonstrated that the HT7 protein is induced in vessels which had vascularized a quail embryonic brain graft transplanted into the coelomic cavity of a chick embryo. Thus, the invading host endothelial cells differentiated into BBB-forming vessels under the influence of developing quail brain cells. As this model seems to reproduce the BBB induction it can be considered useful for other approaches to study the molecular mechanisms involved in BBB induction.

By freeze fracture analysis done in collaboration with Hartwig Wolburg’s laboratory, Werner Risau could demonstrate that P-face association of tight junction particles rather than the number or branching frequency of tight junction strands correlates with BBB function. This suggests that the cytoplasmic anchoring of the tight junction plays an important role in the function of the barrier. Occludin is an integral membrane protein specifically associated with tight junctions. Although it has been demonstrated not to be necessary for the induction of functional tight junctions, it was found here by establishing transfectants with an N-terminally truncated occludin that the N-terminal half of occludin plays an important role in tight junction assembly and maintainance of the barrier function.

Selected reading:
• Wolburg H, Neuhaus J, Kniesel U, Krauss B, Schmid EM, Ocalan M, Farrell C, Risau W. Modulation of tight junction structure in blood-brain barrier endothelial cells. Effects of tissue culture, second messengers and cocultured astrocytes. J Cell Sci 1994 107:1347-1357
• Engelhardt, B., W. Risau. 1995. Development of the blood-brain barrier. "New concepts of a Blood-Brain Barrier". Eds. J. Greenwood, D. Begley, M.Segal , and S. Lightman. Plenum Press, pp. 11-31.
• Bamforth SD, Kniesel U, Wolburg H, Engelhardt B, Risau W. A dominant mutant of occludin disrupts tight junction structure and function. J Cell Sci 1999 112:1879-88
occludin (left) and ZO1 (right) distribution

Werner Risau´s capability to integrate different directions in the field of endothelial cell biology research is exemplified by work that demonstrated that VEGF excerts its vascular permeability function by modulation of junctional proteins. Werner Risau´s latest vision was that the wnt-proteins, which are important regulators in cell and tissue polarity might be involved in the induction or maintenance of the BBB.

Selected reading:
• Esser S, Lampugnani MG, Corada M, Dejana E, Risau W. Vascular endothelial growth factor induces VE-cadherin tyrosine phosphorylation in endothelial cells. J Cell Sci 1998 111:1853-65
• Esser S, Wolburg K, Wolburg H, Breier G, Kurzchalia T, Risau W. Vascular endothelial growth factor induces endothelial fenestrations in vitro. J Cell Biol 1998 Feb 23;140(4):947-59