Specific targeting of gene expression to a subset of human trabecular meshwork cells using the chitinase 3-like 1 promoter

Abstract

Purpose: To compare the gene expression profile of trabecular meshwork (TM) and Schlemm's canal (SC) primary cultures and to identify promoters for targeting gene expression to specific cells in the outflow pathway.

Methods: The differential gene expression profile of four human TM and three SC primary cultures was analyzed by gene microarrays (Affymetrix, Santa Clara, CA) and confirmed by quantitative real-time PCR. Based on the results, a recombinant adenovirus was constructed with the expression of the reporter gene LacZ driven by the 5' promoter region of the chitinase 3-like 1 (Ch3L1) gene (AdCh3L1-LacZ). The expression of the Ch3L1 promoter was analyzed in human TM and SC cells and in human perfused anterior segments infected with AdCh3L1-LacZ.

Results: gamma-Sarcoglycan, fibulin-2, and collagen XV were identified as the genes more highly expressed in SC than in TM cells. Ch3L1 showed the highest levels of differential expression in TM versus SC cells. Expression analysis of the Ch3L1 promoter demonstrated specific expression in a subset of the TM cells in cell culture and in perfused anterior segments.

Conclusions: Comparative analysis of gene expression between SC and TM primary cultures identified several genes with promoters potentially capable of targeting gene expression to specific cells within the outflow pathway. Results with the Ch3L1 promoter indicated that two different cell subtypes may be present in the TM. This study provides a new potential tool to investigate the role of these different cell types in both normal and pathophysiological function of the outflow pathway, with implications for possible future glaucoma gene therapy.

Figure 1

Confirmation of the differential expression between human TM and SC cultured cells by real-time PCR. Expression differences (x-fold) based on the fluorescence threshold values (C_t) calculated using the thermocycler software are presented for three genes with promoters potentially useful for targeting gene expression to TM but not SC cells: Ch3L1, MGP, and CDT6; and three genes with promoters potentially useful for targeting gene expression to SC but not TM cells: COLXV, FBLN2, and SGCG.

Figure 2

Expression analysis of the Ch3L1 promoter. Cells from primary cultures of SC and TM cells and from perfused human anterior segments were infected with the AdCh3L1-LacZ recombinant adenovirus expressing the reporter gene LacZ under the control of a 1059-bp fragment of the Ch3L1 promoter, and analyzed for β-galactosidase expression 48 hours after infection. (A) Primary cultures of human SC cells did not show any detectable expression of the Ch3L1 promoter. (B) Primary cultures of HTM cells showed a mixture of Ch3L1 positive and negative cells indicative of the presence of at least two subpopulations with distinct patterns of gene expression. (C) Control cultures infected with the AdMGP-LacZ virus demonstrated that virtually all the cells in the culture expressed this TM marker. (D) Perfused anterior segments from human eyes showed preferential expression in the TM and in blood vessels of the sclera. (E) Low-magnification view of paraffin sections showed Ch3L1 expression preferentially located in the anterior and posterior regions of the TM. (F) Higher magnification views of paraffin sections also demonstrated the presence of a mixture of positive and negative Ch3L1 cells in the tissue. Similar results including lack of detectable expression in SC cells and heterogeneous staining of TM cells were obtained in both, primary cultures from three different cell lines, and in organ culture from six perfused anterior segments.