Abstract
Purpose: :
Trabecular Meshwork (TM) extracellular matrix (ECM) remodeling and ECM protein deposition are key properties related to intraocular pressure (IOP) elevation, a major glaucoma risk factor. Transforming growth factor-beta 2 (TGF-β2) is elevated in glaucomatous aqueous humor and TM, and induces ECM proteins, including fibronectin (FN), leading to ocular hypertension. However, little is know about the expression of the cellular spliced isoforms of FN (cFN, ED-A or ED-B) and whether TGF-β2 influences FN splicing in the human TM. Therefore, the purpose of this study is to identify the cFN isoform expression in human TM cells and to determine whether TGF-β2 alters FN splicing.
Methods: :
Human TM (HTM) cells were cultured for 0.5, 2, 4 and 7 days in serum reduced medium in the presence or absence of TGF-β2 (5ng/ml). Changes in expression of total FN or cFN isoforms ED-A or ED-B were determined by RT-qPCR analysis. Specific exon PCR primers were used to amplify the relevant splicing domains. Protein content and cellular distribution of ED-A FN were analyzed by immunocytochemistry and by western immunoblotting.
Results: :
All cultured HTM cells expressed both cFN, ED-A and ED-B FN isoforms. Furthermore, TGF-β2 significantly increases mRNA expression of total FN as well as ED-A and ED-B isoforms 1.5-4 fold after 2, 4 and 7 days of treatment (p<0.05). Similarly, TGF-β2 increased ED-A protein expression 3-8 fold (p<0.05) and altered its cellular distribution after 4 and 7 days.
Conclusions: :
Our results present the first evidence of cFN isoforms in human TM cells and the ability of TGF-β2 to alter FN splicing. TGF-β2 modulation of cFN expression in HTM cells suggests a novel mechanism of TM ECM remodeling that may impair TM ECM functions, leading to the IOP elevation associated with glaucoma. Understanding the TM ECM remodeling mechanisms related to glaucoma may lead to the discovery of agents that will normalize TM function and reduce further glaucoma damage.
Keywords: extracellular matrix • trabecular meshwork • cytokines/chemokines