Elucidating the mechanism of action of alpha-1-antitrypsin using retinal pigment epithelium cells exposed to high glucose. Potential use in diabetic retinopathy.

BACKGROUND: Alpha-1-antitrypsin is a protein involved in avoidance of different processes that are seen in diabetic retinopathy pathogenesis. These processes include apoptosis, extracellular matrix remodeling and damage of vessel walls and capillaries. Furthermore, because of its anti-inflammatory effects, alpha-1-antitrypsin has been proposed as a possible therapeutic approach for diabetic retinopathy. Our group tested alpha-1-antitrypsin in a type 1 diabetes mouse model and observed a reduction of inflammation and retinal neurodegeneration. Thus, shedding light on the mechanism of action of alpha-1-antitrypsin at molecular level may explain how it works in the diabetic retinopathy context and show its potential for use in other retinal diseases. METHODS: In this work, we evaluated alpha-1-antitrypsin in an ARPE-19 human cell line exposed to high glucose. We explored the expression of different mediators on signaling pathways related to pro-inflammatory cytokines production, glucose metabolism, epithelial-mesenchymal transition and other proteins involved in the normal function of retinal pigment epithelium by RT-qPCR and Western Blot. RESULTS: We obtained different expression patterns for evaluated mediators altered with high glucose exposure and corrected with the use of alpha-1-antitrypsin. CONCLUSIONS: The expression profile obtained in vitro for the evaluated proteins and mRNA allowed us to explain our previous results obtained on mouse models and to hypothesize how alpha-1-antitrypsin hinder diabetic retinopathy progression on a complex network between different signaling pathways. GENERAL SIGNIFICANCE: This network helps to understand the way alpha-1-antitrypsin works in diabetic retinopathy and its scope of action.

Alpha-1-antitrypsin ameliorates inflammation and neurodegeneration in the diabetic mouse retina.

Diabetic retinopathy (DR) is the most common cause of blindness in the working age population. Early events of DR are accompanied by neurodegeneration of the inner retina resulting in ganglion cell loss. These findings together with reduced retinal thickness are observed within the first weeks of experimental DR. Besides, an inflammatory process is triggered in DR in which the innate immune response plays a relevant role. Alpha 1 antitrypsin (AAT), an inhibitor of serine proteases, has shown anti-inflammatory properties in several diseases. We aimed at evaluating the use of AAT to prevent the early changes induced by DR. Diabetic AAT-treated mice showed a delay on ganglion cell loss and retinal thinning. These animals showed a markedly reduced inflammatory status. AAT was able to preserve systemic and retinal TNF-α level similar to that of control mice. Furthermore, retinal macrophages found in the AAT-treated diabetic mouse exhibited M2 profile (F4/80+CD206+) together with an anti-inflammatory microenvironment. We thus demonstrated that AAT-treated mice show less retinal neurodegenerative changes and have reduced levels of systemic and retinal TNF-α. Our results contribute to shed light on the use of AAT as a possible therapeutic option in DR.

Possible neuroprotective role of P2X2 in the retina of diabetic rats.

BACKGROUND: Purinergic receptors are expressed in different tissues including the retina. These receptors are involved in processes like cell growth, proliferation, activation and survival. ATP is the major activator of P2 receptors. In diabetes, there is a constant ATP production and this rise of ATP leads to a persistent activation of purinergic receptors. Antagonists of these receptors are used to evaluate their inhibition effects. Recently, the P2X2 has been reported to have a neuroprotective role. METHODS: We carried out a study in groups of diabetic and non-diabetic rats (N = 5) treated with intraperitoneal injections of PPADS, at 9 and 24 weeks of diabetes. Control group received only the buffer. Animals were euthanized at 34 weeks of diabetes or at a matching age. Rat retinas were analyzed with immunohistochemistry and western blot using antibodies against GFAP, P2X2, P2Y2 and VEGF-A. RESULTS: Diabetic animals treated with PPADS disclosed a much more extended staining of VEGF-A than diabetics without treatment. A lower protein expression of VEGF-A was found at the retina of diabetic animals without treatment of purinergic antagonists compared to diabetics with the antagonist treatment. Inhibition of P2X2 receptor by PPADS decreases cell death in the diabetic rat retina. CONCLUSION: Results might be useful for better understanding the pathophysiology of diabetic retinopathy.

Corneal Neovascularization: A Combined Approach of Bevacizumab and Suramin Showed Increased Antiangiogenic Effect Through Downregulation of BFGF and P2X2.

PURPOSE: The objective is to analyze the antiangiogenic mechanism of suramab, a pharmaceutical compound of bevacizumab and suramin, in a rabbit model of corneal angiogenesis. MATERIAL AND METHODS: Corneal neovascularization was induced in four groups of six New Zealand White rabbits by applying a filter paper disk soaked in 1 M Na (OH) on the central cornea. Group one was treated after injury with intravenous suramab at a dose equivalent to 3 mg/kg of bevacizumab and 10 mg/kg of suramin. Group two was treated with intravenous bevacizumab (5 mg/kg). Group three was treated with 10 mg/kg of suramin while the control group received no treatment. Digital photographs were taken at days 9, 15, 21, and 35. Neovessel formation was quantified giving a 0-4 score to each quadrant according to the centripetal growth of the longest vessel (neovessel index, NVI). Animals were sacrificed at day 35. Corneas were processed for histology, immunohistochemistry, and Western-blot using primary antibodies against P2X2, basic fibroblast growth factor (bFGF), LYVE-1, PECAM-1, and vascular endothelial growth factor-A (VEGF-A). RESULTS: Suramab significantly reduced neovessel growth (mean NVI: 4.2) compared to bevacizumab (8.4), suramin (7.22), and control animals (12.2) at 35 days post-injury (p < 0.01). A lower protein expression of P2X2, bFGF, LYVE-1, PECAM-1, and VEGF-A was found in the cornea of suramab animals than in the other groups of animals. CONCLUSIONS: Joint downregulation of bFGF, P2X2, bFGF, and LYVE-1 constitutes a mechanism that induces greater and longer inhibition of corneal angiogenesis. Results might be relevant to ophthalmic care. Ocular administration of suramab is currently being investigated.

Suramab, a novel antiangiogenic agent, reduces tumor growth and corneal neovascularization.

PURPOSE: Oncological and ophthalmological diseases are increasingly treated with antiangiogenic agents. These agents have different intensities and duration of effects that should be considered to choose the most suitable therapy. Our purpose was to evaluate the synergistic effect of two drugs, jointly administered as a pharmaceutical compound, in two animal models. METHODS: Corneal neovascularization was induced in three groups of nine white New Zealand rabbits, applying a filter paper disk soaked in 1 M NaOH on the central cornea (Ormerod et al., Invest Ophthalmol Vis Sci 30:2148-2153, 1989). Group one was treated immediately after injury with intravenous Suramab, compound of Bevacizumab + Suramin, and group two with intravenous Bevacizumab. A third group of non-treated rabbits was included as control group. Digital photographs were taken at days 9, 15, 21, and 35. Neovessel index (NVI) was calculated using the Image J Program. Neovessels formation was quantified and given a score from 0 to 4 to each quadrant according to the centripetal growth of the longest vessel. Colorectal animal model: 6- to 8-week-old male BALB/c mice were inoculated with cancer cells. Seven days after tumor inoculation, four groups of BALB/c mice were treated with intravenous Bevacizumab (n = 9); intravenous Suramin (n = 10); intravenous Suramab (n = 10); and intravenous saline solution (n = 4). Tumor growth was assessed twice weekly by caliper measurement. RESULTS: The NVI was remarkably inferior in the group of rabbits treated with intravenous Suramab compared with controls after 35 days of follow-up. A greater inhibitory effect was obtained with Suramab compared to that obtained with Bevacizumab. Suramab significantly reduced tumor volume and prolonged survival of mice compared to controls. CONCLUSIONS: Suramab strongly reduced neovascularization in a rabbit model of corneal angiogenesis and induced a potent antitumoral effect in mice.