Browsing by Author "Pattabiraman, Padmanabhan"

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    Elucidating the Role of Ref-1 in Retinal Neovascularization
    (2025-05) Hartman, Gabriella D.; Yoder, Karmen K.; Corson, Timothy W.; Kelley, Mark R.; Fishel, Melissa L.; Pattabiraman, Padmanabhan
    Retinal neovascularization in retinopathy of prematurity (ROP) and proliferative diabetic retinopathy (PDR) impairs vision. Current treatments of intravitreal (IVT) antivascular endothelial growth factor (VEGF) injections are accompanied by high treatment burden and resistance. Also, VEGF alone is not sufficient for induction of neovascularization, suggesting that targeting multiple disease-relevant pathways may increase therapeutic response. Thus, there is a critical need to develop novel therapies that modulate multiple disease-relevant pathways. APE1/Ref-1, a multifunctional protein with both endonuclease (APE1) and redox regulatory activity (Ref-1), activates multiple transcription factors linked to retinal neovascularization. However, the precise molecular mechanisms of Ref-1 in retinal neovascularization remain unclear. To investigate this, I examined the expression of Ref- 1 in two mouse models of retinal neovascularization – the oxygen-induced retinopathy (OIR) and the Vldlr-/- model – and observed elevated Ref-1 expression at sites of neovascularization. Further, I observed heightened expression of Ref-1 in endothelial cells in human PDR tissue compared to normal eyes. Through cell-based and biochemical approaches, I identified that Ref-1 redox activity modulates canonical Wnt signaling, and inhibition of Ref-1 redox activity blocks Wnt signaling activation. I found that Ref-1 redox activity regulates HIF-1a transcriptional activation in hypoxic human retinal endothelial cells. Hypoxia-induced activation of Wnt signaling was regulated by Ref-1, suggesting a dynamic Ref-1-HIF-1a-Wnt signaling axis. Finally, inhibition of Ref-1 redox activity decreased retinal neovascularization and downregulated expression of Wnt- and angiogenesis-related genes at sites of neovascularization in a mouse model of retinal neovascularization. These findings suggest that Ref-1 redox activity promotes ischemic retinal neovascularization via Wnt signaling activation. This study advanced our understanding of Ref-1’s role in neovascular eye diseases, and targeting Ref-1 with a redox inhibitor could offer a novel therapeutic strategy for retinal neovascularization.
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    Modulation of Intralocular Pressure by Tuning Transcriptional Control of Lipid Synthesis
    (2024-06) Wang, Ting; Morral, Nuria; Pattabiraman, Padmanabhan; Corson, Timothy W.; Landreth, Gary E.; Perrin, Benjamin J.
    Glaucoma is an age-related optic neuropathy and is one of the leading causes of irreversible blindness. Primary open-angle glaucoma (POAG) is the predominant subtype of glaucoma. Elevated intraocular pressure (IOP) is a major risk factor for POAG and lowering IOP is the most effective therapeutic strategy. IOP is maintained by the balance of aqueous humor (AH) generation by the ciliary body and drainage by conventional outflow pathway including trabecular meshwork (TM). TM is a highly contractile and mechanosensitive tissue, and its contractility regulated by the actin cytoskeleton and extracellular matrix (ECM) is directly related to IOP regulation. Using multiomics analysis in human TM (HTM) cells, I identified that mechanical stretch caused the activation of sterol regulatory element binding proteins (SREBPs) related-lipid biogenesis pathways. Further, using immunofluorescence, and constitutive activation of each SREBP isoform, I discovered the mechanosensing role of SREBPs in HTM cells and mechanistically deciphered the attributes of SREBPs in regulating the contractile properties of TM. The pharmacological inhibition of SREBPs by fatostatin and molecular inactivation of SREBPs ex vivo and in vivo resulted in significant IOP lowering. Conversely, significantly elevated IOP was observed after using the pharmacological activator of SREBPs by clozapine and constitutive activation of SREBPs ex vivo and in vivo, respectively. As a proof of concept, fatostatin significantly decreased the SREBPs responsive genes and enzymes involved in lipogenic pathways and phospholipids, cholesterol, and triglyceride levels. The increased lipid biogenesis was found after constitutive activation of SREBP isoforms in HTM cells but with slightly different effects between each isoform. Further, I showed that fatostatin mitigated actin polymerization machinery and stabilization, and identified that SREBPs activation is a critical regulator of ECM engagement to the matrix sites. Lastly, I identified that cholesterol levels play an important role in regulating actin polymerization, focal adhesion formation, cell-ECM interactions, and membrane tension in HTM cells. Therefore, we have established the direct connection between cholesterol and TM contractility. Overall, I postulate that lowering de novo lipogenesis in the TM outflow pathway can hold the key to lowering IOP by modifying the TM biomechanics.