Superior Properties of N-Acetylcysteine Ethyl Ester over N-Acetyl Cysteine to Prevent Retinal Pigment Epithelial Cells Oxidative Damage.

Oxidative stress plays a key role in the pathophysiology of retinal diseases, including age-related macular degeneration (AMD) and diabetic retinopathy, which are the major causes of irreversible blindness in developed countries. An excess of reactive oxygen species (ROS) can directly cause functional and morphological impairments in retinal pigment epithelium (RPE), endothelial cells, and retinal ganglion cells. Antioxidants may represent a preventive/therapeutic strategy and reduce the risk of progression of AMD. Among antioxidants, N-acetyl-L-cysteine (NAC) is widely studied and has been proposed to have therapeutic benefit in treating AMD by mitigating oxidative damage in RPE. Here, we demonstrate that N-acetyl-L-cysteine ethyl ester (NACET), a lipophilic cell-permeable cysteine derivative, increases the viability in oxidative stressed RPE cells more efficiently than NAC by reacting directly and more rapidly with oxidizing agents, and that NACET, but not NAC, pretreatment predisposes RPE cells to oxidative stress resistance and increases the intracellular reduced glutathione (GSH) pool available to act as natural antioxidant defense. Moreover, we demonstrate the ability of NACET to increase GSH levels in rats' eyes after oral administration. In conclusion, even if experiments in AMD animal models are still needed, our data suggest that NACET may play an important role in preventing and treating retinal diseases associated with oxidative stress, and may represent a valid and more efficient alternative to NAC in therapeutic protocols in which NAC has already shown promising results.

Publisher Correction: TGF-ß concentrations and activity are down-regulated in the aqueous humor of patients with neovascular age-related macular degeneration.

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TGF-ß concentrations and activity are down-regulated in the aqueous humor of patients with neovascular age-related macular degeneration.

Controversy still exists regarding the role of the TGF-ß in neovascular age-related macular degeneration (nAMD), a major cause of severe visual loss in the elderly in developed countries. Here, we measured the concentrations of active TGF-ß1, TGF-ß2, and TGF-ß3 by ELISA in the aqueous humor of 20 patients affected by nAMD, who received 3 consecutive monthly intravitreal injections of anti-VEGF-A antibody. Samples were collected at baseline (before the first injection), month 1 (before the second injection), and month 2 (before the third injection). The same samples were used in a luciferase-based reporter assay to test the TGF-ß pathway activation. Active TGF-ß1 concentrations in the aqueous humor were below the minimum detectable dose. Active TGF-ß2 concentrations were significantly lower at baseline and at month 1, compared to controls. No significant differences in active TGF-ß3 concentration were found among the sample groups. Moreover, TGF-ß pathway activation was significantly lower at baseline compared to controls. Our data corroborate an anti-angiogenic role for TGF-ß2 in nAMD. This should be considered from the perspective of a therapy using TGF-ß inhibitors.

Dissecting the CD93-Multimerin 2 interaction involved in cell adhesion and migration of the activated endothelium.

The glycoprotein CD93 has recently been recognized to play an important role in the regulation of the angiogenic process. Moreover, CD93 is highly expressed in the endothelial cells of tumor blood vessel and faintly expressed in the non-proliferating endothelium. Much evidence suggests that CD93 mediates adhesion in the endothelium. Here we identify Multimerin 2 (MMRN2), a pan-endothelial extracellular matrix protein, as a specific ligand for CD93. We found that CD93 and MMRN2 are co-expressed in the blood vessels of various human tumors. Moreover, disruption of the CD93-MMRN2 interaction reduced endothelial cell adhesion and migration, making the interaction of CD93 with MMRN2 an ideal target to block pathological angiogenesis. Model structures and docking studies served to envisage the region of CD93 and MMRN2 involved in the interaction. Site-directed mutagenesis identified different residue hotspots either directly or indirectly involved in the binding. We propose a molecular model in which the coiled-coil domain of MMRN2 is engaged by F238 of CD93. Altogether, these studies identify the key interaction surfaces of the CD93-MMRN2 complex and provide a framework for exploring how to inhibit angiogenesis by hindering the CD93-MMRN2 interaction.

HTRA1 and TGF-ß1 Concentrations in the Aqueous Humor of Patients With Neovascular Age-Related Macular Degeneration.

Purpose: The purpose of this study was to evaluate the expression of high-temperature requirement A serine peptidase 1 (HTRA1), TGF-ß1, bone morphogenetic protein 4 (BMP4), growth differentiation factor 6 (GDF6), and VEGFA proteins in the aqueous humor of patients with naïve choroidal neovascularization (nCNV) secondary to AMD. Methods: We measured by ELISA the concentrations of HTRA1, TGF-ß1, BMP4, GDF6, and VEGFA in the aqueous humor of 23 patients affected by nCNV who received three consecutive monthly intravitreal injections of 0.5 mg ranibizumab. Samples were collected at baseline (before the first injection), month 1 (before the second injection), and month 2 (before the third injection). Twenty-three age-matched cataract patients served as controls. Results: Bone morphogenetic protein 4 and GDF6 were not detectable in any samples. Baseline HTRA1 was higher than controls (P < 0.0001) and higher than both the month 1 (P < 0.0001) and the month 2 (P < 0.0001) values. Baseline VEGFA was higher than controls (P < 0.0001), not different from month 1 value (P = 0.0821), but higher than month 2 value (P < 0.0001). Baseline TGF-ß1 was higher than controls (P = 0.0015) and not different from month 1 (P = 0.129) and month 2 values (P = 0.5529). No correlation was found in naïve patients between concentrations of HTRA1 and TGF-ß1, HTRA 1 and VEGFA, or TGF-ß1 and VEGFA. Conclusions: In nCNV patients, HTRA1 and TGF-ß1 were significantly higher compared to controls. After treatment, TGF-ß1 was persistently elevated, while HTRA1 returned to control levels, suggesting the involvement of TGF-ß1 and HTRA1 in neovascular AMD and a VEGFA-independent role for TGF-ß1.

CD93 as a Potential Target in Neovascular Age-Related Macular Degeneration.

In patients with age-related macular degeneration (AMD), choroidal neovascularization is the major cause of severe visual loss. In these patients, the persistence of neovascular growth despite vascular endothelial growth factor-A blockage needs the discovery of new endothelial cell targets. The glycoprotein CD93, highly expressed in activated endothelial cells, has been recently involved in the regulation of the angiogenic process both as transmembrane and soluble protein. Choroidal neovascular membranes from patients affected by AMD were examined by immunofluorescence using anti-CD93 and anti-von Willebrand factor antibodies. Blood vessels within intraocular and extraocular neoplasias were used as controls for CD93 expression. All choroidal neovascular membranes displayed strong CD93 staining in the von Willebrand factor-positive endothelial cells, consistently with the analyses showing a high colocalization coefficient in the blood vessels. Intraocular and extraocular tumor vessels showed similar results, whereas the normal choroid displayed blood vessels with only faint CD93 staining. Additionally, the concentration of soluble CD93 was determined in the aqueous humor of patients affected by naïve neovascular AMD by enzyme-linked immunosorbent assays. Age-matched cataract patients served as controls. Soluble CD93 was significantly increased in the aqueous humor of naïve neovascular AMD patients and tended to decrease after treatment with an antiangiogenic drug. In conclusion, both transmembrane and soluble CD93 are overexpressed in patients with neovascular AMD, indicating that CD93 may represent a potential new antiangiogenic target in the treatment of choroidal neovascularization. J. Cell. Physiol. 232: 1767-1773, 2017. © 2016 Wiley Periodicals, Inc.