TGFβ2-Hepcidin Feed-Forward Loop in the Trabecular Meshwork Implicates Iron in Glaucomatous Pathology

Abstract

Elevated levels of transforming-growth-factor (TGF)-β2 in the trabecular meshwork (TM) and aqueous humor are associated with primary open-angle glaucoma (POAG). The underlying mechanism includes alteration of extracellular matrix homeostasis through Smad-dependent and independent signaling. Smad4, an essential co-Smad, upregulates hepcidin, the master regulator of iron homeostasis. Here, we explored whether TGF-β2 upregulates hepcidin, implicating iron in the pathogenesis of POAG. METHODS. Primary human TM cells and human and bovine ex vivo anterior segment organ cultures were exposed to bioactive TGF-β2, hepcidin, heparin (a hepcidin antagonist), or N-acetyl carnosine (an antioxidant), and the change in the expression of hepcidin, ferroportin, ferritin, and TGF-β2 was evaluated by semiquantitative RT-PCR, Western blotting, and immunohistochemistry. Increase in reactive oxygen species (ROS) was quantified with dihydroethidium, an ROS-sensitive dye. RESULTS. Primary human TM cells and bovine TM tissue synthesize hepcidin locally, which is upregulated by bioactive TGF-β2. Hepcidin downregulates ferroportin, its downstream target, increasing ferritin and iron-catalyzed ROS. This causes reciprocal upregulation of TGF-β2 at the transcriptional and translational levels. Heparin downregulates hepcidin, and reduces TGF-β2-mediated increase in ferritin and ROS. Notably, both heparin and N-acetyl carnosine reduce TGF-β2-mediated reciprocal upregulation of TGF-β2. CONCLUSIONS. The above observations suggest that TGF-β2 and hepcidin form a selfsustained feed-forward loop through iron-catalyzed ROS. This loop is partially disrupted by a hepcidin antagonist and an anti-oxidant, implicating iron and ROS in TGF-β2mediated POAG. We propose that modification of currently available hepcidin antagonists for ocular use may prove beneficial for the therapeutic management of TGF-β2-associated POAG.

Retinal Degeneration and Alzheimer’s Disease: An Evolving Link

“…In the brain, hepcidin is expressed in the cortex, hippocampus, cerebellum, thalamus, and medulla oblongata [ 108 , 109 , 110 ]. In the eye, the synthesis and expression of hepcidin is noted in several cell types in the retina and the anterior segment [ 111 , 112 , 113 ].…”

“…In a recent report, the upregulation of hepcidin in TM cells by TGFβ2 initiated a positive feed-forward loop between TGFβ2, hepcidin, and iron fueled by ROS. Disruption of this loop with hepcidin antagonists and antioxidants reduced the iron accumulation and ROS, suggesting a prominent role of ROS in primary open-angle glaucoma [ 111 , 118 ] ( Figure 3 ). In addition, significant protection of RGCs is achieved by chelating iron, reinforcing the toxic role of iron in glaucomatous degeneration [ 119 ].…”

“…These agents, in addition to reducing the hepcidin-mediated accumulation of iron, are likely to reduce inflammation as well, adding to their therapeutic potential. In an in vitro model of glaucoma, the cytokine-mediated upregulation of hepcidin was disrupted with heparin, a hepcidin antagonist, and N-acetyl L carnosine (NAC), an antioxidant [ 111 ], suggesting that such agents are useful in decreasing the iron-catalyzed ROS and may prove useful in rescuing neurons and RGCs from ROS-mediated toxicity and death. Inflammatory changes accompany glaucomatous degeneration [ 131 ], and the use of anti-inflammatory compounds, including nonsteroidal anti-inflammatory drugs (NSAID) such as pranoprofen, have been useful in decreasing the IOP and rescue RGCs [ 133 ].…”

Iron chelators: as therapeutic agents in diseases

“…Hepcidin is found in various brain areas, including the cortex, hippocampus, cerebellum, thalamus, and medulla oblongata 125 – 127 . In the eye, hepcidin is synthesized and expressed in different cell types in the retina and front part of the eye 128 – 130 . Although hepcidin helps maintain strict control over iron levels, it can be increased by inflammatory signals such as IL-6, IL-1β, and transforming growth factors β1 and β2.…”

Prions and prion diseases: Insights from the eye

“…Autocrine and paracrine activity of hepcidin downregulates Fpn, resulting in intracellular accumulation of iron, compensatory upregulation of ferritin, and increase in ROS, which upregulates TGFβ2. Heparin, a hepcidin antagonist, and antioxidants inhibit upregulation of TGFβ2 by decreasing ROS, disrupting the cycle (Ashok et al, 2020a). Butaux et al, 2015;Ashok et al, 2019;Kim et al, 2020).…”