Caio V. Regatieri

Heparan sulfate proteoglycans: structure, protein interactions and cell signaling

Heparan sulfate proteoglycans are ubiquitously found at the cell surface and extracellular matrix in all the animal species. This review will focus on the structural characteristics of the heparan sulfate proteoglycans related to protein interactions leading to cell signaling. The heparan sulfate chains due to their vast structural diversity are able to bind and interact with a wide variety of proteins, such as growth factors, chemokines, morphogens, extracellular matrix components, enzymes, among others. There is a specificity directing the interactions of heparan sulfates and target proteins, regarding both the fine structure of the polysaccharide chain as well precise protein motifs. Heparan sulfates play a role in cellular signaling either as receptor or co-receptor for different ligands, and the activation of downstream pathways is related to phosphorylation of different cytosolic proteins either directly or involving cytoskeleton interactions leading to gene regulation. The role of the heparan sulfate proteoglycans in cellular signaling and endocytic uptake pathways is also discussed.

CHOROIDAL IMAGING USING SPECTRAL-DOMAIN OPTICAL COHERENCE TOMOGRAPHY

Background: A structurally and functionally normal choroidal vasculature is essential for retinal function. Therefore, a precise clinical understanding of choroidal morphology should be important for understanding many retinal and choroidal diseases. Methods: PUBMED (http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed) was used for most of the literature search for this article. The criterion for inclusion of an article in the references for this review was that it included materials about both the clinical and the basic properties of choroidal imaging using spectral-domain optical coherence tomography. Results: Recent reports show successful examination and accurate measurement of choroidal thickness in normal and pathologic states using spectral-domain optical coherence tomography systems. This review focuses on the principles of the new technology that make choroidal imaging using optical coherence tomography possible and on the changes that subsequently have been documented to occur in the choroid in various diseases. Additionally, it outlines future directions in choroidal imaging. Conclusion: Optical coherence tomography is now proven to be an effective noninvasive tool to evaluate the choroid and to detect choroidal changes in pathologic states. Additionally, choroidal evaluation using optical coherence tomography can be used as a parameter for diagnosis and follow-up.

Therapeutic monoclonal antibodies in ophthalmology.

Monoclonal antibodies (mAbs) can be used therapeutically by binding to molecular targets with high specificity. Therefore, they have excellent therapeutic applications in ophthalmology. This manuscript presents four aspects of the therapeutic use of mAbs in ophthalmology: the scientific rationale, the unique characteristics of selected mAbs, the current state-of-the-art application, and relevant therapeutic mAbs for future applications in ophthalmology. We identified in the literature various single-agent therapies that inhibit the following targets: tumor necrosis factor (TNF), epithelial growth factor receptor, vascular endothelial growth factor (VEGF) receptor, basic fibroblast growth factor receptor, platelet-derived growth factor, and cluster of differentiation antigens. The roles of all biochemical targets in ocular diseases were evaluated. Current and future mAbs against various cytokines were assessed for the treatment of ocular diseases. The medical literature showed the clinical benefits of mAbs for treating angiogenic and inflammatory ocular diseases. Two anti-VEGF mAbs, bevacizumab and ranibizumab, and three anti-TNF agents, infliximab, etanercept, and adalimumab, control ocular neovascularization and intraocular inflammation. Other mAbs such as rituximab, daclizumab, efalizumab, and alemtuzumab showed positive results in animal and early clinical studies and may represent useful adjuvant therapies for ocular lymphoma or ocular inflammation. Ranibizumab is the only FDA-approved therapy; for other mAbs the so-called off-label application remains the standard. Intravenous administration of mAbs has demonstrated acceptable toxicity profiles, while intraocular injection may decrease the chances of systemic complications and increase the amount of drug available to the retina and choroid. In conclusion, effective clinical use of mAbs in ophthalmology is more commonly seen in the field of angiogenic vitreoretinal and autoimmune inflammatory diseases. The challenge for the future is combining biologic therapies to improve the quality and duration of responses while diminishing side effects. The role of mAbs within ophthalmic treatments will be defined according to future clinical experience and the results of randomized clinical trials.

Retinal and Ocular Toxicity in Ocular Application of Drugs and Chemicals – Part II: Retinal Toxicity of Current and New Drugs

Aims: Retinal pharmacotherapy has gained great importance for the treatment of various retinal diseases. An increasing number of drugs have been constantly released into the market, especially for wet age-related macular disease and diabetic macular edema. In this review, the issues concerning the toxicity of current and new classes of drugs are discussed. Methods: An extensive search of the literature was performed to review various aspects of drug toxicity in retinal pharmacotherapy. The different major classes of drugs, such as corticosteroids, antibiotics, antimetabolites, antineoplastic agents, monoclonal antibodies (mAbs), nonsteroidal anti-inflammatory drugs, enzymes, fibrinolytics, miscellaneous anti-inflammatory and antiangiogenic agents, as well as toxicity unrelated to the drug were identified and discussed. Results: Corticosteroids like fluocinolone, dexamethasone or triamcinolone at low dose cause little damage to the retina, but at high doses signs of toxicity have been well documented. Complications like cataract and glaucoma are quite common with corticosteroids. Aminoglycosides showed differences in the type and doses associated with toxic reactions, thereby the following order of toxicity can be described (from most toxic to least toxic): gentamicin > netilmicin = tobramycin > amikacin = kanamycin. Vancomycin at the usual dose of 1 mg is not toxic to the retina, while further studies are necessary in order to clarify the safety of new-generation quinolones. 5-Fluorouracil has been shown to be nontoxic to the retina after an injection of 2.5 mg in animals. mAbs like ranibizumab and bevacizumab were demonstrated to be safe to the retina in cell culture, animals and humans at high doses. The exact biocompatibility of nonsteroidal anti-inflammatory agents like diclofenac needs further evaluation. Preservatives like benzyl alcohol and changes in pH or osmolarity exert an influence on the toxic effects of intravitreally applied drugs. Conclusions: A great number of drugs are now used mainly intravitreally without relevant retinal toxicity.

Growth inhibition and pro-apoptotic activity of violacein in Ehrlich ascites tumor

The continuing threat to biodiversity lends urgency to the need of identification of sustainable source of natural products. This is not so much trouble if there is a microbial source of the compound. Herein, violacein, a natural indolic pigment extracted from Chromobacterium violaceum, was evaluated for its antitumoral potential against the Ehrlich ascites tumor (EAT) in vivo and in vitro. Evaluation of violacein cytotoxicity using different endpoints indicated that EAT cells were twofold (IC(50)=5.0 microM) more sensitive to the compound than normal human peripheral blood lymphocytes. In vitro studies indicated that violacein cytotoxicity to EAT cells is mediated by a rapid (8-12h) production of reactive oxygen species (ROS) and a decrease in intracellular GSH levels, probably due to oxidative stress. Additionally, apoptosis was primarily induced, as demonstrated by an increase in Annexin-V positive cells, concurrently with increased levels of DNA fragmentation and increased caspase-2, caspase-9 and caspase-3 activities up to 4.5-, 6.0- and 5.5-fold, respectively, after 72 h of treatment. Moreover, doses of 0.1 and 1.0 microg kg(-1) violacein, administered intraperitoneally (i.p.) to EAT-bearing mice throughout the lifespan of the animals significantly inhibited tumor growth and increased survival of mice. In view of these results, a 35-day toxicity study was conducted in vivo. Complete hematology, biochemistry (ALT, AST and creatinine levels) and histopathological analysis of liver and kidney indicated that daily doses of violacein up to 1000 microg kg(-1) for 35 days are well tolerated and did not cause hematotoxicity nor renal or hepatotoxicity when administered i.p. to mice. Altogether, these results indicate that violacein causes oxidative stress and an imbalance in the antioxidant defense machinery of cells culminating in apoptotic cell death. Furthermore, this is the first report of its antitumor activity in vivo, which occurs in the absence of toxicity to major organs.

Dual Role of Intravitreous Infliximab in Experimental Choroidal Neovascularization: Effect on the Expression of Sulfated Glycosaminoglycans

PURPOSE To determine effects of intravitreous anti-TNF-alpha (infliximab) in a laser-induced choroidal neovascularization (CNV) model by fluorescein angiogram (FA), immunofluorescence, ELISA, and glycosaminoglycan analyses. METHODS CNV induction was performed using argon laser. Rats were divided into eight groups (no-laser no-infliximab; laser; laser with 10, 20, 40, 80, or 320 microg infliximab; and isotype-matched IgG). After 3 weeks, CNV area was measured by FA and von Willebrand factor (vWF) immunofluorescence. VEGF, TGF-beta, and syndecan-4 were evaluated by ELISA and immunofluorescence. Glycosaminoglycan expression was determined in retina and choroid of animals metabolically labeled with [(35)S]-sulfate. Cytotoxicity was investigated using ARPE-19 and endothelial cells. RESULTS FA showed significant reduction in the low-dose infliximab groups (10-40 microg), confirmed by vWF immunofluorescence that showed 49% decrease in the CNV. VEGF and TGF-beta decreased expression detected by ELISA and immunofluorescence paralleled these results. Similar data were observed for syndecan-4. The expression of these molecules in the neovascularization area using 320 microg was similar to the no-infliximab laser group or laser with isotype-matched IgG. Heparan sulfate expression in retina and choroid paralleled the observed effects on angiogenesis. Increased expression of chondroitin sulfate in retina and dermatan sulfate in choroid reflects the effects of injury and fibrosis using high doses of anti-TNF-alpha. Infliximab showed no cytotoxic effect in ARPE-19 cells, whereas high doses led to 20% decrease in endothelial cell viability. CONCLUSIONS Intravitreal infliximab shows dual effect on the development of laser-induced CNV. It reduces angiogenesis and glycosaminoglycan expression at low doses, whereas opposite effects are observed at high doses.

Effect of Intravitreous Anti–Vascular Endothelial Growth Factor Therapy on Choroidal Thickness in Neovascular Age-Related Macular Degeneration Using Spectral-Domain Optical Coherence Tomography

A critical method of monitoring patients with neovascular age-related macular degeneration (AMD) being treated with anti–vascular endothelial growth factor (anti-VEGF) is optical coherence tomography (OCT), which uses low-coherence interferometry of light to examine the retina in vivo on a micrometer scale.1 Recent advances in spectral-domain OCT make visualization of the choroid feasible. Using image averaging and enhanced depth imaging, successful examination and measurement of choroidal thickness in normal and pathologic states have been reported.2–4 It has been hypothesized that anti-VEGF may affect choroidal vasculature.5 The goal of this study is to evaluate the effect of anti-VEGF on choroidal thickness using spectral-domain OCT in treatment-naive subjects.

The Role of Spectral-Domain OCT in the Diagnosis and Management of Neovascular Age-Related Macular Degeneration

Spectral-domain optical coherence tomography (SD-OCT) has emerged as the ancillary examination of choice to assist the diagnosis and management of neovascular age-related macular degeneration (AMD). SD-OCT provides more detailed images of intraretinal, subretinal, and subretinal pigment epithelium fluid when compared to time-domain technology, leading to higher and earlier detection rates of neovascular AMD activity. Improvements in image analysis and acquisition speed make it important for decision-making in the diagnosis and treatment of this disease. However, this new technology needs to be validated for its role in the improvement of visual outcomes in the context of anti-angiogenic therapy.

Evaluation of spectral domain and time domain optical coherence tomography findings in toxoplasmic retinochoroiditis

Purpose: To investigate spectral domain optical coherence tomography (SD-OCT) findings and compare them with time domain (TD)-OCT imaging of macula and retinochoroiditis lesions of patients with toxoplasmosis. Design: Prospective cross-sectional study. Methods: Ten eyes of 10 patients with active toxoplasmic retinochoroiditis were included. Morphologic features from the macula and retinochoroiditis lesions were obtained at baseline and at 6-week follow up. Scan acquisition protocols for TD-OCT included raster and radial lines through the retinochoroiditis lesion, fast macular, and a linear scan from the lesion to the fovea, whereas the acquisition protocols for SD-OCT also included horizontal volume scans at the lesion site and at the macula. Thickness measurements obtained by SD-OCT were analyzed. Results: At baseline, macular serous retinal detachment was observed in five patients; two of them only seen by SD-OCT. Retinochoroidal lesions were 4260 μm distant from the fovea on average (R = 681–7130) and this distance had an indirect correlation to the presence of macular detachment. Epiretinal membrane and vitreo-macular traction were also observed. The posterior hyaloid was not identified in four patients by TD-OCT and only in one by SD-OCT at baseline. Perilesional subretinal fluid was observed in two patients. The median retinal thickness significantly decreased at the retinochoroiditis lesion (P = 0.0004), and all the patients remained with disorganized retinal layers reflectivity at follow up. Conclusion: SD-OCT is a useful tool in the diagnosis of macular changes related with toxoplasmic retinochoroiditis. SD-OCT is superior in evaluating retinal changes associated with ocular toxoplasmosis.

Alkali burn to the eye: protection using TNF-α inhibition.

Purpose: The aim of this study was to evaluate early retinal damage after induction of ocular surface alkali burns and the protective effects of tumor necrosis factor alpha (TNF-&agr;) blockade. Methods: Alkali injury was induced in mouse corneas by using 1 N NaOH. Retinal damage was assessed using a terminal deoxynucleotidyl transferase 2′-deoxyuridine 5-triphosphate nick end labeling (TUNEL) assay, 15 minutes to 14 days postburn. Immune cell infiltration was assessed by CD45 immunolocalization. Retinal cytokines were quantified using the enzyme-linked immunosorbent assay for interleukin (IL)1&bgr;, IL2, IL6, TNF-&agr;, CCL5, and macrophage inflammatory protein-1&agr;. Protection against retinal damage was attempted with a single dose of either anti–TNF-&agr; antibody (infliximab, 6.25 mg/kg) or control immunoglobulin G (IgG), administered intraperitoneally 15 minutes after the burn was inflicted. Corneal injury was evaluated by using TUNEL and CD45 immunolocalization and by quantifying corneal neovascularization. Results: There was significant damage to the retina within 24 hours of the corneal burn being inflicted. TUNEL+ labeling was present in 80% of the retinal ganglion cells, including a few CD45+ cells. There was a 10-fold increase in the retinal inflammatory cytokines in the study groups compared with that in controls. A single intraperitoneal dose of anti–TNF-&agr; antibody, administered 15 minutes after the burn, markedly reduced retinal TUNEL+, CD45+ labeling, and inflammatory cytokine expression, compared with that in the controls. Additionally, TNF-&agr; blockade caused a marked reduction in corneal neovascularization, and in cornea TUNEL and CD45 labeling, 5 days after the burn. Conclusions: This study shows that alkali corneal burns can induce significant retinal damage within 24 hours. A single dose of anti–TNF-&agr; antibody, administered 15 minutes after inflicting the burn, provides significant retinal and corneal protection. This could lead to the discovery of novel therapies for patients with alkali injuries.