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PATOS Research Center

Pathophysiology and Treatment of Stroke

Project – Dr. Bickel

The long-term goal of this project is to minimize brain damage, which develops secondary to the inflammatory reactions occurring in the wake of brain ischemia/reperfusion. Brain microvascular endothelial cells (the “blood-brain barrier” ) are at the center of this inflammatory response. We pursue a targeted delivery approach for drugs, such as oligodeoxynucleotide “decoys” to the transcription factor NF- k B , to brain endothelial cells. These drugs exert anti-inflammatory effects by inhibiting the gene expression of mediators and of cell adhesion molecules , which attract circulating cells of the immune system to the site of ischemic brain injury. Delivery strategies for oligodeoxy-nucleotides and other DNA-based drugs using complexes with cationic polymers and encapsulation in liposomes are developed and tested both in cell culture models ( Figure 1 ) and in vivo.

We have shown that monocyte adhesion to brain-derived endothelial cells in vitro can be significantly inhibited by such an approach ( Figure 2 ).

Figure 1 shows the time dependent cellular uptake of a fluorescent labeled oligodeoxy-nucleotide decoy by endothelial cells. The ODN is bound by a cationic polymer, forming a complex of about 100 nm size, which is targeted to cell surface receptors by a monoclonal antibody (transferring receptor antibody 8D3). These receptors mediate uptake by the cells.

Figure 2 shows a quantitative assay for adhesion of fluorescent labeled monocytes (cell line U-937) to brain-derived endothelial cells (EC). The transcription factor decoy (ODN) was applied at 2 μM concentration with different cationic polymers ( PEI). There was little adhesion to non-stimulated EC. The “media control” shows the percentage of added monocytes adhering to EC after inflammatory stimulation with endotoxin. “Total monocytes” equals 100% of monocytes added per culture well (= reference)