Increasing endothelial cell permeability improves the efficiency of myocyte adenoviral vector infection.
BACKGROUND
Gene delivery to the myocardium using blood-borne adenoviral vectors is hindered by the endothelium, which represents a barrier limiting the infection rate of underlying myocytes. However, endothelial permeability may be modulated by pharmacological agents.


METHODS
In the present study, we modeled the endothelial barrier in vitro using a human umbilical vein endothelial cell (HUVEC) monolayer seeded on a Transwell membrane as a support and diffusion of fluorescent dextrans as a permeability index. We used alpha-thrombin (100 nM) as a pharmacological agent known to increase endothelial permeability and tested the barrier function of the endothelial cell monolayer on adenovector-mediated luciferase gene transfer to underlying isolated cardiac myocytes.


RESULTS
A confluent HUVEC monolayer represented a considerable physical barrier to dextran diffusion; it reduced the permeability of the micropore membrane alone to fluorescein isothiocyanate (FITC)-labeled dextrans of molecular weights 4, 70, 150 and 2000 kDa by approximately 54, 78, 88 and 98%, respectively. Alpha-thrombin (100 nM) increased the permeability coefficients (P(EC)) by 276, 264, 562 and 4166% for the same dextrans, respectively. A confluent HUVEC monolayer represented a major impediment to adenovector-mediated luciferase gene transfer to cardiac myocytes, largely reducing gene transfer efficiency. However thrombin induced a nine-fold increase in myocyte infection.


CONCLUSIONS
In our model, the endothelial cell monolayer represents a major impediment to myocyte adenovector-mediated gene transfer which can be partially improved by pharmacologically increasing endothelial permeability. The Transwell model is therefore particularly useful for testing the efficiency of pharmacological agents in modulating adenovector passage through the endothelial barrier.
Nevo
N
N
INSERM U460, Faculté de Médecine Xavier Bichat, Paris, France. nevo@bichat.inserm.fr
Chossat
N
N
Gosgnach
W
W
Logeart
D
D
Mercadier
J J
JJ
Michel
J B
JB
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002199:Capillary Permeability / Q000187:drug effects; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013917:Thrombin / Q000494:pharmacology
D013917:Thrombin