Elevated intraocular pressure (IOP) is a major risk factor for the development and progression of primary open angle glaucoma and is due to trabecular meshwork (TM) damage, which leads to impaired aqueous humor outflow. Here, we explore a novel molecular mechanism involved in glaucomatous TM damage. We investigated the role of an endogenous Toll-like receptor 4 (TLR4) ligand, fibronectin-EDA (FN-EDA), in TGFβ2-induced ocular hypertension in mice. We utilized transgenic mouse strains that either constitutively express only FN containing the EDA isoform or contain an EDA-null allele and express only FN lacking EDA, with or without a mutation in Tlr4, in our inducible mouse model of ocular hypertension by injection of Ad5.TGFβ2. IOP was measured over time and eyes accessed by immunohistochemistry for total FN and FN-EDA expression. Constitutively active EDA caused elevated IOP starting at 14 weeks of age. Ad5.TGFβ2 induced ocular hypertension in wildtype C57BL/6J mice and further amplified the IOP in constitutively active EDA mice. TLR4 null and EDA null mice blocked Ad5.TGFβ-induced ocular hypertension. Total FN and FN-EDA isoform expression increased in response to Ad5.TGFβ2. These data suggest that both TLR4 and FN-EDA contribute to TGFβ2 induced ocular hypertension.
PurposeThe trabecular meshwork (TM) has an important role in the regulation of aqueous humor outflow and IOP. Regulation of the extracellular matrix (ECM) by TGFβ2 has been studied extensively. Bone morphogenetic protein (BMP) and activin membrane-bound inhibitor (BAMBI) has been shown to inhibit or modulate TGFβ2 signaling. We investigate the role of TGFβ2 and BAMBI in the regulation of TM ECM and ocular hypertension.MethodsMouse TM (MTM) cells were isolated from B6;129S1-Bambitm1Jian/J flox mice, characterized for TGFβ2 and dexamethasone (DEX)–induced expression of fibronectin, collagen-1, collagen-4, laminin, α-smooth muscle actin, cross-linked actin networks (CLANs) formation, and DEX-induced myocilin (MYOC) expression. MTM cells were transduced with Ad5.GFP to identify transduction efficiency. MTM cells and mouse eyes were transduced with Ad5.Null, Ad5.Cre, Ad5.TGFβ2, or Ad5.TGFβ2 + Ad5.Cre to evaluate the effect on ECM production, IOP, and outflow facility.ResultsMTM cells express TM markers and respond to DEX and TGFβ2. Ad5.GFP at 100 MOI had the highest transduction efficiency. Bambi knockdown by Ad5.Cre and Ad5.TGFβ2 increased fibronectin, collagen-1, and collagen-4 in TM cells in culture and tissue. Ad5.Cre, Ad5.TGFβ2, and Ad5.TGFβ2 + Ad5.Cre each significantly induced ocular hypertension and lowered aqueous humor outflow facility in transduced eyes.ConclusionsWe show for the first time to our knowledge that knockdown of Bambi alters ECM expression in cultured cells and mouse TM, reduces outflow facility, and causes ocular hypertension. These data provide a novel insight into the development of glaucomatous TM damage and identify BAMBI as an important regulator of TM ECM and ocular hypertension.
Spaceflight-Associated Neuro-ocular Syndrome (SANS) is a significant unexplained adverse reaction to long-duration spaceflight. We employ an ex vivo translaminar autonomous system (TAS) to recreate a human ocular ground-based spaceflight analogue model to study SANS pathogenesis. To recapitulate the human SANS conditions, human ocular posterior segments are cultured in the TAS model for 14 days. Translaminar pressure differentials are generated by simulating various flow rates within intracranial pressure (ICP) and intraocular (IOP) chambers to maintain hydrostatic pressures of ICP: IOP (12:16, 15:16, 12:21, 21:16 mmHg). In addition, optic nerves are mechanically kinked by 6- and 10-degree tilt inserts for the ICP: IOP;15:16 mmHg pressure paradigm. The TAS model successfully maintains various pressure differentials for all experimental groups over 14 days. Post culture, we determine inflammatory and extracellular component expression changes within posterior segments. To further characterize the SANS pathogenesis, axonal transport capacity, optic nerve degeneration and retinal functional are measured. Identifiable pathogenic alterations are observed in posterior segments by morphologic, apoptotic, and inflammatory changes including transport and functional deficits under various simulated SANS conditions. Here we report our TAS model provides a unique preclinical application system to mimic SANS pathology and a viable therapeutic testing device for countermeasures.
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