Retroviral gene transfer: effects on endothelial cell phenotype.
BACKGROUND
Endothelial cells (EC) are an attractive target for somatic cell gene therapy, both for the treatment of cardiovascular disease and for the systemic delivery of recombinant gene products directly into the circulation. Recent evidence, however, suggests that viral transduction may induce unfavorable changes in EC phenotype. We examined the proliferative capacity and cell adhesion molecule (CAM) profile of EC after retroviral gene transfer (GT), employing a clinically relevant ex vivo GT protocol.


METHODS
Human umbilical vein EC (HUVEC, N = 14 isolates) were exposed to supernatants containing the MFG.nlsLACZ vector, which codes for a nuclear localized beta-galactosidase. Control HUVEC were exposed to empty virus (CRIP) or no virus (NT). Efficiency of GT was quantitated by direct counting of beta-galactosidase-stained cells on a grid. Proliferation was quantitated by a 1-week assay of viable cell counts. Expression of EC activation molecules (Class II major histocompatibility antigen [MHC II], E-selectin, intercellular adhesion molecule-1 [ICAM-1], and vascular cell adhesion molecule-1 [VCAM-1]) was examined using fluorescent cytometry (FACS) at rest and after cytokine stimulation.


RESULTS
GT was reproducibly efficient (mean 57%, range 40-77%) using sequential viral exposures without selection. NT, CRIP, and LACZ-transduced HUVEC exhibited identical FACS profiles for E-selectin, ICAM-1, VCAM-1, and MHC II at rest, consistent with a nonactivated state. Upregulation of expression by cytokine was quantitatively similar for all groups. Growth rates were likewise not different between groups.


CONCLUSIONS
Retroviral vectors may be employed to achieve high percentages of transduced EC for ex vivo GT without the use of selection. Transduced EC generated in this fashion are not activated, demonstrate an unaltered pattern of inducible CAM expression, and exhibit normal cell growth. The effects of GT on target cell phenotype are likely to be both vector and protocol specific and should be carefully assessed in each case prior to in vivo applications.
Inaba
M
M
Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut, USA.
Toninelli
E
E
Vanmeter
G
G
Bender
J R
JR
Conte
M S
MS
D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D019040:E-Selectin / Q000032:analysis / Q000235:genetics; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology*; D018014:Gene Transfer Techniques; D000949:Histocompatibility Antigens Class II / Q000032:analysis / Q000235:genetics; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000032:analysis / Q000235:genetics; D007763:Lac Operon; D009052:Leukemia Virus, Murine; D010641:Phenotype; D014161:Transduction, Genetic; D014471:Umbilical Veins / Q000166:cytology; D019010:Vascular Cell Adhesion Molecule-1 / Q000032:analysis / Q000235:genetics
D019040:E-Selectin; D000949:Histocompatibility Antigens Class II; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1