Abstract
Abstract: :
Purpose: To investigate whether human trabecular meshwork cells (HTM) undergo calcification. To determine the conformational status of the Matrix Gla protein (MGP), an inhibitor of soft tissue calcification, in the human TM. Methods: Primary HTM cell lines were derived from individuals with no history of glaucoma. Vitamin K–dependent γ–carboxylase activity was measured as 14C–γ–carboxylation of the peptide substrate FLEEL. Senescence cells were obtained by incubating late–passage cells for 4–5 weeks. Calcification was assessed by alizarin red staining. An adenoviral vector encoding the MGP protein under the control of a CMV promoter (AdhMGP) was constructed by bacterial transposition, LacZ disruption selection and 293 cells plasmid transfection. Human anterior segments were perfused and infected by standard methods. Western blots were performed with two types of antibodies: MGP–Gla, that recognizes the mature, functional protein and MGP–N that recognizes MGP independently of its conformational (γ–carboxylation) status. Results:HTM cell calcification (alizarin red staining) increases markedly with age, with old cells forming "calcification nodules". HTM cells have significant carboxylase activity. Endogenous MGP in the active, carboxylated state was easily detected in perfused TM and hardly detected in primary HTM cells; however the HTM cells were able to readily carboxylate gene transferred recombinant MGP. Delivery of recombinant MGP was very efficient and dose dependent. Conclusions: The human trabecular meshwork possesses a previously undescribed calcification mechanism. Consistent with calcification of the arterial system, the HTM cells appear to also calcify with aging. MGP, a regulator of soft tissue calcification, is very abundant in the TM and can be converted to its conformational active form. Delivery of recombinant MGP could potentially be used in the future to modulate and protect trabecular meshwork calcification. J.L.V. current affiliation: Inspire Pharmaceuticals , Inc., Durham, NC.
Keywords: trabecular meshwork • protein modifications–post translational • aging