Fibronectin extra domain A (FN-EDA) elevates intraocular pressure through Toll-like receptor 4 signaling.

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 TGFß2-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.TGFß2. 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.TGFß2 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.TGFß-induced ocular hypertension. Total FN and FN-EDA isoform expression increased in response to Ad5.TGFß2. These data suggest that both TLR4 and FN-EDA contribute to TGFß2 induced ocular hypertension.

Nuclear factor-kappa beta signaling is required for transforming growth factor Beta-2 induced ocular hypertension.

A major risk for the development of primary open-angle glaucoma (POAG) is elevated intraocular pressure (IOP). Elevated IOP is caused by increased outflow resistance due in part to excessive extracellular matrix (ECM) deposition in the trabecular meshwork (TM). The role of transforming growth factor beta 2 (TGFß2) in inducing ECM production is well understood. Recent studies suggest that toll-like receptor 4 (TLR4) plays an important role in fibrogenesis. We have previously described a crosstalk between TGFß2 and TLR4 in the development of ocular hypertension and glaucomatous TM damage. Nuclear factor-kappa beta (NF-κB) is critical for TLR4 signaling. To determine the transactivation of NF-κB, TM cells were stimulated with cellular fibronectin containing the EDA isoform (cFN-EDA), TGFß2, or lipopolysaccharide (LPS) in combination with a selective TLR4 inhibitor. cFN-EDA, TGFß2, and LPS all induced transactivation of NF-κB and inhibition of TLR4 blocked the effect of each treatment paradigm. To evaluate the role of NF-κB in IOP regulation, we utilized our inducible mouse model of ocular hypertension by injection of Ad5.TGFß2 in mice harboring a mutation in NF-κB and wild-type controls. IOP was measured over time and eyes accessed by immunohistochemistry for the ECM protein FN and the specific FN-EDA isoform. Ad5.TGFß2 induced ocular hypertension and expression of FN and FN-EDA in wild-type mice, but mutation in NF-κB blocked the effect. These data suggest that NF-κB is necessary for TGFß2-induced ECM production and ocular hypertension and the transactivation of NF-κB is dependent on both TGFß2 and TLR4.

Corticotropin-releasing factor receptors CRF1 and CRF2 exert both additive and opposing influences on defensive startle behavior.

The corticotropin-releasing factor (CRF) receptors (CRF1 and CRF2) are crucial mediators of physiological and behavioral responses to stress. In animals, CRF1 appears to primarily mediate CRF-induced anxiety-like responses, but the role of CRF2 during stress is still unclear. Here we report the effects of CRF1 and CRF2 on the magnitude and plasticity of defensive startle responses in mice. Startle plasticity is measured by inhibition of startle by sensory stimuli, i.e., prepulse inhibition (PPI), and is disrupted in patients with panic or posttraumatic stress disorders in which CRF neurotransmission may be overactive. Pharmacological blockade of CRF1 reversed both CRF-induced increases in startle and CRF-induced deficits in PPI. CRF2 blockade attenuated high-dose but not low-dose CRF-induced increases in startle and reduced PPI. Conversely, activation of CRF2 enhanced PPI. CRF had no effect on startle and increased PPI in CRF1 knock-out mice. These data indicate that CRF receptors act in concert to increase the magnitude of defensive startle yet in opposition to regulate the flexibility of startle. These data support a new model of respective CRF receptor roles in stress-related behavior such that, although both receptors enhance the magnitude of defensive responses, CRF1 receptors contravene, whereas CRF2 receptors enhance, the impact of sensory information on defensive behavior. We hypothesize that excessive CRF1 activation combined with reduced CRF2 signaling may contribute to information processing deficits seen in panic and posttraumatic stress disorder patients and support CRF1-specific pharmacotherapy.