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Anita W. Krysta, Matthias Zenkel, Marco Birke, Anselm Jünemann, Gabriele Gusek-Schneider, Friedrich E. Kruse, Ursula Schlotzer-Schrehardt; In Vitro Model For The Functional Analysis Of LOXL1 Gene Variants In The Pathophysiology Of Pseudoexfoliation Syndrome. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3852.
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© ARVO (1962-2015); The Authors (2016-present)
Lysyl oxidase-like 1 (LOXL1) gene variants have been identified as the most significant genetic risk factor for pseudoexfoliation (PEX) syndrome, a complex disorder of the elastic fiber system and frequent cause of glaucoma. To evaluate the role of LOXL1, an elastin cross-linking enzyme, in the pathophysiology of PEX syndrome/glaucoma, we developed a cell culture model for the functional analysis of PEX-associated risk variants in extracellular matrix formation.
Haplotypes of the non-synonymous LOXL1 variants rs1048661 and rs3825942 were generated by site-directed mutagenesis of full-length human LOXL1 cDNA and cloned into the mammalian expression vector pCMV-Entry. Human Tenon’s capsule fibroblasts (hTCF) were isolated from normal donor eyes and transfected with the pCMV-LOXL1 constructs by nucleofection (Amaxa). To select stably transfected clones cells were grown in DMEM/F12 medium containing 30% FCS and 800µg/ml G418 for 2 weeks. Extracellular matrix synthesis was stimulated by TGF-ß1. Expression profiles of LOXL1, BMP-1 and elastic fiber constituents (elastin, fibrillin-1, fibulin-4, fibulin-5) were analyzed in cell lysates and media up to 14 days post-confluence using Real-time PCR, Western blotting, and light and electron microscopic immunocytochemistry; LOXL1 activity was assessed by a high sensitive fluorescent assay.
Transfection efficiency ranged from 15-20% in transiently transfected to 80% in stably transfected hTCF cells. Transfected cells could be expanded as stably transgene-expressing clones over 3 weeks enabling long-term analysis of elastic fiber assembly. Stable clones produced a high steady-state level of LOXL1, which was 300-fold increased compared to non-transfected controls and could be localized to cytoplasmic secretory vesicles. TGF-ß1 predominantly stimulated co-expression of elastic proteins and promoted incorporation of elastin into a preformed microfibrillar scaffold 10-14 days post-confluence. LOXL1, together with its processing protease BMP-1, was secreted into culture medium and was found to associate with elastic fibers in the extracellular space.
The results demonstrate the efficiency of nucleofected primary fibroblasts as a model system to analyze synthesis, secretion and targeting of LOXL1 to the extracellular elastic matrix. This cell-based assay may represent a useful strategy to unravel critical functional differences of LOXL1 gene variants predisposing to PEX syndrome and glaucoma.
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