Technical Brief Determination of active TGF-β2 in aqueous humor prior to and following cryopreservation

Transforming growth factor-β 2 (TGF-β 2 ) is an approximately 25 kDa polypeptide encoded by a unique gene located on chromosome 1q41 [1]. This dimeric peptide is ubiquitously distributed in human tissues and synthesized by many different human cells. It has been detected in tear fluid, in the vitreous, and in aqueous humor [2-4]. TGF-β 2 is known to play an important role in wound healing and the production of the extracellular matrix. It inhibits cell proliferation and exerts various immunosuppressive effects. In the anterior chamber, TGF-β 2 is relevant for the maintenance of an immunosuppressive climate, as it alters the activities of antigen-presenting cells, and suppresses T-cell proliferation, IFN-γ production, and the inflammatory activity of macrophages [4-6]. It is secreted as an inactive precursor (latent TGF-β 2 complex, LTGF-β 2 ) by cell types such as corneal endothelial cells, cells of the trabecular meshwork, and the ciliary body [7,8]. This precursor (200 kDa) is complexed with latency-associated peptide (LAP) and bound to latent TGF-β binding protein (LTBP). L-TGF-β 2 is not able to bind to its receptor until LAP and LTBP are removed extracellularly via proteolytic cleavage. The exact mechanisms by which latent TGF-β 2 is activated physiologically are not completely understood. One model of activation has been proposed in which latent TGF-β is released from the extracellular matrix by proteases, localized to cell surfaces, and activated for example by thrombospondin-1 [9] or specific integrins [10]. Following this activation, TGF-β 2 exerts its biological functions via binding to a membrane-bound heteromeric receptor [8]. In addition to TGF-β 2 two further isoforms of TGF-β have been identified in mammals, TGF-β 1 and TGF-β 3 . Both isoforms play an only minor role in immunomodulation of the anterior ocular segment [11]. Different levels of total TGF-β 2 have been found in human aqueous humor, depending on the ocular disorders concerned [12,13]. The ratio of active to total TGF-β 2 in the aqueous humor has been reported to be between 11 to 61% [1419]. However, in those studies the concentration of active TGF©2006 Molecular Vision