Expression analysis of the matrix GLA protein and VE-cadherin gene promoters in the outflow pathway.
OBJECTIVE
To test the ability of promoter fragments from the matrix Gla protein (MGP) and vascular endothelial-cadherin (VE-cad) genes to target gene expression in a specific manner in the cells of the outflow pathway, by using adenoviral-mediated gene transfer in organ culture.


METHODS
Perfused anterior segments of human eyes were infected with replication-deficient recombinant adenoviruses expressing the beta-galactosidase reporter gene driven by the cytomegalovirus (CMV; control, n = 6), MGP (n = 6), or VE-cad (n = 12) promoters. Forty-eight hours after infection, the anterior segments were fixed and stained for beta-galactosidase activity. The distribution of beta-galactosidase expression was analyzed in paraffin-embedded sections.


RESULTS
The MGP promoter fragment resulted in beta-galactosidase expression by the cells of the conventional outflow pathway and did not show any activity in the corneal endothelium or other cells posterior to the scleral spur. Adenovirus containing the VE-cad promoter fragment showed functionality of the promoter in vascular endothelial cells, but failed to produce any detectable expression in the cells of the outflow pathway.


CONCLUSIONS
Directed expression by the MGP gene promoter specifically to the trabecular meshwork (TM) provides a new tool for specific gene transfer to the outflow pathway. Results with the VE-cad promoter fragment indicate possible differences in the regulation of this gene between vascular and Schlemm's canal endothelial cells. Taken together, these data demonstrate the feasibility of targeted gene expression to the outflow pathway cells using tissue specific promoters.
Gonzalez
Pedro
P
Department of Ophthalmology, Duke University, Durham, North Carolina 27710, USA. pedro.gonzalez@duke.edu
Caballero
Montserrat
M
Liton
Paloma B
PB
Stamer
W Daniel
WD
Epstein
David L
DL
D000260:Adenoviruses, Human / Q000235:genetics; D000328:Adult; D000368:Aged; D015703:Antigens, CD; D015820:Cadherins / Q000235:genetics* / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D016326:Extracellular Matrix Proteins; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D008875:Middle Aged; D009924:Organ Culture Techniques; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014129:Trabecular Meshwork / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism; D000097568:Matrix Gla Protein; D000099152:Cadherin 5
D015703:Antigens, CD; D015820:Cadherins; D002135:Calcium-Binding Proteins; D016326:Extracellular Matrix Proteins; D001616:beta-Galactosidase; D000097568:Matrix Gla Protein; D000099152:Cadherin 5