TY - JOUR
T1 - Genetic transfer of fusion proteins effectively inhibits VCAM-1-mediated cell adhesion and transmigration via inhibition of cytoskeletal anchorage
AU - Hagemeyer, Christoph Eugen
AU - Ahrens, Ingo
AU - Bassler, Nicole
AU - Dschachutaschwili, Natia
AU - Chen, Yung-Chih
AU - Eisenhardt, Steffen U
AU - Bode, Christoph
AU - Peter, Karlheinz
PY - 2010
Y1 - 2010
N2 - The adhesion of leukocytes to endothelium plays a central role in the development of atherosclerosis and thus represents an attractive therapeutic target for anti-atherosclerotic therapies. Vascular cell adhesion molecule-1 (VCAM-1) mediates both the initial tethering and the firm adhesion of leukocytes to endothelial cells. Our work evaluates the feasibility of using the cytoskeletal anchorage of VCAM-1 as a target for gene therapy. As a proof of concept, integrin aIIb?3-mediated cell adhesion with clearly defined cytoskeletal anchorage was tested. We constructed fusion proteins containing the intracellular domain of ?3 placed at various distances to the cell membrane. Using cell adhesion assays and immunofluorescence, we established fusion constructs with competitive and dominant negative inhibition of cell adhesion. With the goal being the transfer of the dominant negative mechanism towards VCAM-1 inhibition, we constructed a fusion molecule containing the cytoplasmic domain of VCAM-1. Indeed, VCAM-1 mediated leukocyte adhesion can be inhibited via transfection of DNA encoding the designed VCAM-1 fusion protein. This is demonstrated in adhesion assays under static and flow conditions using CHO cells expressing recombinant VCAM-1 as well as activated endothelial cells. Thus, we are able to describe a novel approach for dominant negative inhibition of leukocyte adhesion to endothelial cells. This approach warrants further development as a novel gene therapeutic strategy that aims for a locally restricted effect at atherosclerotic areas of the vasculature.
AB - The adhesion of leukocytes to endothelium plays a central role in the development of atherosclerosis and thus represents an attractive therapeutic target for anti-atherosclerotic therapies. Vascular cell adhesion molecule-1 (VCAM-1) mediates both the initial tethering and the firm adhesion of leukocytes to endothelial cells. Our work evaluates the feasibility of using the cytoskeletal anchorage of VCAM-1 as a target for gene therapy. As a proof of concept, integrin aIIb?3-mediated cell adhesion with clearly defined cytoskeletal anchorage was tested. We constructed fusion proteins containing the intracellular domain of ?3 placed at various distances to the cell membrane. Using cell adhesion assays and immunofluorescence, we established fusion constructs with competitive and dominant negative inhibition of cell adhesion. With the goal being the transfer of the dominant negative mechanism towards VCAM-1 inhibition, we constructed a fusion molecule containing the cytoplasmic domain of VCAM-1. Indeed, VCAM-1 mediated leukocyte adhesion can be inhibited via transfection of DNA encoding the designed VCAM-1 fusion protein. This is demonstrated in adhesion assays under static and flow conditions using CHO cells expressing recombinant VCAM-1 as well as activated endothelial cells. Thus, we are able to describe a novel approach for dominant negative inhibition of leukocyte adhesion to endothelial cells. This approach warrants further development as a novel gene therapeutic strategy that aims for a locally restricted effect at atherosclerotic areas of the vasculature.
UR - http://onlinelibrary.wiley.com/doi/10.1111/j.1582-4934.2008.00409.x/abstract;jsessionid=7B04BACEE7A87B2D449DBA3E8D6387CB.f04t02
U2 - 10.1111/j.1582-4934.2008.00409.x
DO - 10.1111/j.1582-4934.2008.00409.x
M3 - Article
SN - 1582-1838
VL - 14
SP - 290
EP - 302
JO - Journal of Cellular and Molecular Medicine
JF - Journal of Cellular and Molecular Medicine
IS - 1-2
ER -