Giamberto Casini

Quercetin decreases proliferation of orbital fibroblasts and their release of hyaluronic acid

Background: Inhibition of fibroblast (FB) proliferation and hyaluronic acid (HA) production may be a therapeutic approach to Graves’ ophthalmopathy (GO). The flavonoid quercetin has a wide range of activities, including reduction of FB growth. Aim: To investigate the effects of quercetin in orbital FB from GO patients and control subjects. Methods: Primary cultures of orbital FB were treated with quercetin or with its glycosides rutin and quercitrin. Cell proliferation, necrosis, apoptosis, HA production, and cell cycle were measured. Results: Beginning at a 30 µM concentration, quercetin, but not rutin and quercitrin, reduced cell proliferation, with no difference between GO and control FB. The effect of quercetin on proliferation was due to necrosis and cell cycle blockade, whereas apoptosis was unaffected. Quercetin reduced HA in the cell media, with no difference between GO and control FB. Conclusions: Quercetin reduces cell proliferation and HA release in orbital FB. Whether these initial findings have any potential for the use of quercetin in the clinical practice remains to be established.

INVERTED INTERNAL LIMITING MEMBRANE FLAP TECHNIQUE FOR MACULAR HOLE SURGERY WITHOUT EXTRA MANIPULATION OF THE FLAP

Purpose: To determine whether surgical manipulation steps of the internal limiting membrane (ILM) flap, such as ILM trimmed, ILM tuck inside the hole, ILM massage, are mandatory to obtain satisfactory outcomes for the repair of large stage IV idiopathic macular hole using the inverted ILM flap technique. Methods: In this interventional comparative prospective single-masked study, 81 eyes were randomized into 2 treatments groups. In Group 1 (41 eyes), the classic inverted ILM flap technique was performed. In Group 2 (40 eyes), a modified procedure was used: after ILM peeling, no extra flap manipulation was performed. The macular hole was covered by the inverted ILM flap because of the air pressure at the time of the fluid–air exchange. Results: At 12 months, macular hole closure was observed in 40 eyes (97.6%) in Group 1 and in 39 eyes in Group 2 (97.5%). U-shape closure rate, ellipsoid zone defects, and external limiting membrane defects were similar in both groups. The results indicate no statistical difference in anatomical and functional success between both groups. Conclusion: The macular hole closure rate, improved visual acuity, and no extra complications indicate noninferiority of the modified inverted ILM technique. Internal limiting membrane finishing, tucking, and massage may not be required to obtain surgical success.

IL-8 and VEGFR-2 polymorphisms modulate long-term functional response to intravitreal ranibizumab in exudative age-related macular degeneration.

AIM To investigate possible associations between VEGFR-2 and IL-8 gene SNPs and 1-year response to intravitreal ranibizumab for exudative age-related macular degeneration. MATERIALS & METHODS Sixty-four eyes underwent a loading phase of three monthly intravitreal injections of ranibizumab 0.5 mg/0.05 ml followed by Pro Re Nata retreatment. VEGFR-2 rs2071559 (-604 A/G) and IL-8 rs4073 (-251 A/T) were analyzed. RESULTS Ranibizumab was significantly more effective as measured by visual acuity in patients harboring the IL-8 rs4073 TT genotype (p = 0.045), whereas patients carrying the VEGFR-2 rs2071559 CC genotype revealed better functional response as measured by mean retinal sensitivity (p = 0.034). CONCLUSION IL-8 rs4073 and VEGFR-2 rs2071559 genotypes may represent important molecular determinants to modulate final outcomes in neovascular age-related macular degeneration patients.

Action of three bioavailable antioxidants in orbital fibroblasts from patients with Graves? orbitopathy (GO): a new frontier for GO treatment?

ObjectiveOxidative stress is involved in the pathogenesis of Graves’ orbitopathy (GO) and an antioxidant approach has been advocated for GO treatment. Here, we investigated the action of three antioxidants in orbital fibroblasts, namely, vitamin C, N-acetyl-l-cysteine, and melatonin.MethodsPrimary cultures of orbital fibroblasts from six GO patients and six control subjects were established. Cells were treated with H2O2 to induce oxidative stress. Cell vitality assays were performed to determine the non-cytotoxic dose of each antioxidant. The following assays were performed: glutathione disulfide (GSSG), as a measure of oxidative stress, cell proliferation, hyaluronic acid (HA), TNFα, IFNγ, and IL1β.ResultsH2O2 induced oxidative stress (augmented GSSG), increased cell proliferation as well as cytokine release, but did not affect HA release. All of the three antioxidant substances reduced H2O2-dependent oxidative stress. Vitamin C reduced proliferation in GO, but not in control fibroblasts. N-acetyl-l-cysteine reduced proliferation and IFNγ in GO, and HA and IL1β in both GO and control fibroblasts. Melatonin reduced IL1β and HA in GO and control fibroblasts, and IFNγ only in GO fibroblasts.ConclusionsOur study provides evidence in support of an antioxidant role of vitamin C, N-acetyl-l-cysteine and melatonin in orbital fibroblasts. Some of the effects of these compounds are exclusive to GO fibroblasts, whereas some other are observed also in control fibroblasts. Our observations provide a basis for a possible clinical use of these substances in patients with GO.

Scleral Flap-Everting Suture for Glaucoma-filtering Surgery.

Purpose:Most filtering surgery failures develop in the early postoperative period. The possibility to apply an everting suture to lift the flap in the postoperative period and reduce the possibility of an early failure is reported. Methods:Surgical technique description. Results:An everting suture may be applied to the scleral flap in all types of ab externo anterior filtering surgeries. It could allow the surgeon to lift the scleral flap after the removal of the releasable sutures. Conclusions:The use of an everting suture would eliminate the need to use procedures for lifting the flap that involve puncturing the conjunctiva and may cause hemorrhages and leakage and promote scarring.

Graves’ orbitopathy, idiopathic orbital inflammatory pseudotumor and Epstein–Barr virus infection: a serological and molecular study

AbstractObjectiveOne of the hypotheses on the pathogenesis of autoimmune diseases, including Graves’ disease (GD) and Graves’ orbitopathy (GO), involves bacterial or viral infections. Recently, Epstein–Barr virus (EBV) has been proposed to play a role in the pathogenesis of idiopathic orbital inflammatory pseudotumor (IOIP) in Asians. The aim of the present study was to investigate the possible association of GO with EBV infection/exposure, as compared with IOIP, using serum and tissue samples, as well as primary cultures of orbital fibroblasts.MethodsThirty-one patients were studied, including four with IOIP, ten with GO, nine with GD without GO and eight control patients without IOIP, GD and GO. All patients with IOIP and GO underwent orbital decompression. Control patients underwent palpebral surgery. Fibroadipose orbital tissue samples were collected. Serum anti-EBV antibodies were measured in all patients. EBV-DNA was measured in blood samples, orbital tissue samples and primary cultures of orbital fibroblasts. ResultsSerum assays showed that the vast majority of patients have had a previous exposure to EBV, but no one had an acute infection. EBV-DNA was detected in ~40% of blood samples from GO, GD and control patients, but in none of the IOIP samples. EBV-DNA was not detected in any of the orbital tissue samples tested or in primary cultures of orbital fibroblasts.ConclusionsEBV infection does not seem to be associated with GD, GO and IOIP in Caucasians. Whether EBV is involved in IOIP in Asians or other populations remains to be confirmed.

Selenium rescues orbital fibroblasts from cell death induced by hydrogen peroxide: another molecular basis for the effects of selenium in graves’ orbitopathy

In vivo and in vitro studies suggest that the anti-oxidant agent selenium can be used for the treatment of Graves’ disease (GD) and Graves’ orbitopathy (GO) [1–3]. GO, the most common extrathyroidal manifestation of GD [4–6], is due to an autoimmune reaction against orbital fibroadipose tissue, following which a number of effector mechanisms, including oxidative stress, are responsible for the pathological changes of the orbit [7]. Treatment with selenium is associated with an improvement of mild GO [2], and recently we reported that selenium exerts antioxidant actions in vitro, namely in orbital fibroblasts exposed to oxidative stress by incubation with hydrogen peroxide (H2O2) [3]. H2O2 was cytotoxic if used at concentrations ≥10 μM, but at 5 μM it elicited proliferation of fibroblasts and cytokine release [3]. The effects of H2O2 were counteracted by selenium, which also caused a reduced release of hyaluronic acid [3]. When performing the experiments of our previous study, we observed casually that selenium seemed to counteract also the cytotoxic action of H2O2. Hence, we conducted an additional study aimed at investigating whether selenium is actually capable of inhibiting H2O2-dependent cytotoxicity, as well as at comprehending the mechanisms underlying H2O2 cytotoxicity and selenium actions.

Anti-Vascular Endothelial Growth Factors Protect Retinal Pigment Epithelium Cells Against Oxidation by Modulating Nitric Oxide Release and Autophagy

Background/Aims: the anti-vascular endothelial growth factors (VEGF), Aflibercept and Ranibizumab, are used for the treatment of macular degeneration. Here we examined the involvement of nitric oxide (NO), mitochondria function and of apoptosis/autophagy in their antioxidant effects in human retinal pigment epithelium cells (RPE). Methods: RPE were exposed to Ranibizumab/Aflibercept in the absence or presence of NO synthase (NOS) inhibitor and of autophagy activator/blocker, rapamicyn/3-methyladenine. Specific kits were used for cell viability, NO and reactive oxygen species detection and mitochondrial membrane potential measurement, whereas Western Blot was performed for apoptosis/ autophagy markers and other kinases detection. Results: In RPE cultured in physiological conditions, Aflibercept/Ranibizumab increased NO release in a dose and time-dependent way. Opposite results were obtained in RPE pretreated with hydrogen peroxide. Moreover, both the anti-VEGF agents were able to prevent the fall of cell viability and of mitochondrial membrane potential. Those effects were reduced by the NOS inhibitor and 3-methyladenine and were potentiated by rapamycin. Finally, Aflibercept and Ranibizumab counteracted the changes of apoptosis/autophagy markers, NOS, Phosphatidylinositol-3-Kinase/Protein Kinase B and Extracellular signal–regulated kinases 1/2 caused by peroxidation. Conclusion: Aflibercept and Ranibizumab protect RPE against peroxidation through the modulation of NO release, apoptosis and autophagy.

Emerging surgical therapy in the treatment of glaucoma.

There is general consensus that surgery gives a better intraocular pressure (IOP) control than medical therapy, but surgery may be affected by complications and failures, and for this reason nowadays, it is reserved to advanced or clearly progressive glaucoma. In recent years, there have been a lot of efforts to enhance safety and efficacy of conventional surgery as to find new techniques more safer and more effective. Actually, this is a field in rapid evolution, and we have a great number of innovative procedures, often working on complete different basis. These procedures are classified according to their mechanism of action and the type of surgical approach, in order to clearly understand of what we are speaking about. From a general point of view, surgical procedures may be divided in procedures that increase outflow and procedures that reduce aqueous production: most of these procedures can be performed with an ab externo or an ab interno approach. The ab interno approach has great advantages and enormous potential of development; probably, its diffusion will be facilitated by the development of new devices for angle visualization. Nevertheless, it is important to remember that actually none of the new procedures has been validated in large controlled clinical trials and none of the new procedures is indicated when IOP target is very low.

Antioxidant effects of β-carotene, but not of retinol and vitamin E, in orbital fibroblasts from patients with Graves’ orbitopathy (GO)

BackgroundOxidative stress is involved in the pathogenesis of Graves’ orbitopathy (GO) and several antioxidant agents, namely, selenium, quercetin, enalapril, vitamin C, N-acetyl-l-cysteine, and melatonin, have been shown to reduce oxidative stress and its consequences in primary culture of orbital fibroblasts. In addition, selenium is effective for the treatment of mild GO. Here, we investigated the action of three additional antioxidants in orbital fibroblasts, namely, retinol, β-carotene, and vitamin E.MethodsPrimary cultures of orbital fibroblasts were established from GO patients and control subjects. To induce oxidative stress, cells were treated with H2O2, after which glutathione disulfide (GSSG) (a parameter of oxidative stress), cell proliferation, hyaluronic acid, TNFα, IFNγ, and IL1β were measured.ResultsH2O2-dependent oxidative stress (augmented GSSG) was associated with increased cell proliferation and cytokine release. All the three antioxidant substances reduced GSSG in both GO and control fibroblasts. β-carotene reduced proliferation in GO, but not in control fibroblasts. IL1β was reduced by all three substances. Retinol reduced IFNγ in GO and control fibroblasts.ConclusionsOur study supports an antioxidant role of retinol, β-carotene, and vitamin E in orbital fibroblasts from patients with GO and provides a basis for a possible clinical use these substances.