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.

Transglutaminase binding fusion protein linked to SLPI reduced corneal inflammation and neovascularization.

BACKGROUND: To study the effect of topical administration of a fusion protein (PF-MC) made up of N-terminal portion of the protease inhibitor Trappin-2 (which is a substrate of transglutaminasa-2) and SLPI (protein with anti-inflammatory, anti-bacterial and anti-viral ability), in an animal model of corneal inflammation and angiogenesis. METHODS: An alkali injury was produced with a filter paper of 3 mm with 1 N NaOH during 40 seconds on the right cornea of 36 male Sprague Dawley rats, under general anesthesia. Animals were divided into three groups according to treatment. Group 1 was treated with 10 ul of PF-MC (200 ug/ml; n = 12), Group 2, with 10 ul of SLPI (200 ug/ml; n = 12) and Group 3 was treated with buffer (10 ul; n = 12) topically administered four times a day for up to 7 days. Half of the animals were sacrificed at day 3 before making a re-epithelialization time analysis with fluorescein staining at 18 and 24 hours. In the remaining animals corneal opacity was studied and digital photographs were taken at day 7 before doing euthanasia. Eyes were processed for histology and immunofluorescence. RESULTS: Corneal ulcerated area was significantly lower in PF-MC treated animals compared to SLPI and buffer-treated animals at 18 hours and 24 hours postinjury. A clear cornea and fundus red reflex was only found among PF-MC treated animals. Histological analysis revealed a stratified corneal epithelium with at least three layers in all PF-MC animals at day 7. In this group there was a reduced number of PMNs in the corneal stroma at 3 and 7 days of follow-up. Besides, corneal neovascularization was much more extended in SLPI and Buffer animals than in animals treated with PF-MC. CONCLUSIONS: The binding of SLPI with Cementoin to transglutaminase seems to be an effective strategy to treat corneal inflammation and angiogenesis.

Diabetic retinopathy: could the alpha-1 antitrypsin be a therapeutic option?

Diabetic retinopathy is one of the most important causes of blindness. The underlying mechanisms of this disease include inflammatory changes and remodeling processes of the extracellular-matrix (ECM) leading to pericyte and vascular endothelial cell damage that affects the retinal circulation. In turn, this causes hypoxia leading to release of vascular endothelial growth factor (VEGF) to induce the angiogenesis process. Alpha-1 antitrypsin (AAT) is the most important circulating inhibitor of serine proteases (SERPIN). Its targets include elastase, plasmin, thrombin, trypsin, chymotrypsin, proteinase 3 (PR-3) and plasminogen activator (PAI). AAT modulates the effect of protease-activated receptors (PARs) during inflammatory responses. Plasma levels of AAT can increase 4-fold during acute inflammation then is so-called acute phase protein (APPs). Individuals with low serum levels of AAT could develop disease in lung, liver and pancreas. AAT is involved in extracellular matrix remodeling and inflammation, particularly migration and chemotaxis of neutrophils. It can also suppress nitric oxide (NO) by nitric oxide sintase (NOS) inhibition. AAT binds their targets in an irreversible way resulting in product degradation. The aim of this review is to focus on the points of contact between multiple factors involved in diabetic retinopathy and AAT resembling pleiotropic effects that might be beneficial.

Diabetic retinopathy: could the alpha-1 antitrypsin be a therapeutic option?

Diabetic retinopathy is one of the most important causes of blindness. The underlying mechanisms of this disease include inflammatory changes and remodeling processes of the extracellular-matrix (ECM) leading to pericyte and vascular endothelial cell damage that affects the retinal circulation. In turn, this causes hypoxia leading to release of vascular endothelial growth factor (VEGF) to induce the angiogenesis process. Alpha-1 antitrypsin (AAT) is the most important circulating inhibitor of serine proteases (SERPIN). Its targets include elastase, plasmin, thrombin, trypsin, chymotrypsin, proteinase 3 (PR-3) and plasminogen activator (PAI). AAT modulates the effect of protease-activated receptors (PARs) during inflammatory responses. Plasma levels of AAT can increase 4-fold during acute inflammation then is so-called acute phase protein (APPs). Individuals with low serum levels of AAT could develop disease in lung, liver and pancreas. AAT is involved in extracellular matrix remodeling and inflammation, particularly migration and chemotaxis of neutrophils. It can also suppress nitric oxide (NO) by nitric oxide sintase (NOS) inhibition. AAT binds their targets in an irreversible way resulting in product degradation. The aim of this review is to focus on the points of contact between multiple factors involved in diabetic retinopathy and AAT resembling pleiotropic effects that might be beneficial.