Towards preventing exfoliation glaucoma by targeting and removing fibrillar aggregates associated with exfoliation syndrome.

Exfoliation syndrome presents as an accumulation of insoluble fibrillar aggregates that commonly correlates with age and causes ocular complications, most notably open-angle glaucoma. Despite advances in understanding the pathogenesis and risk factors associated with exfoliation syndrome, there has been no significant progress in curative pharmacotherapy of this disease. It is thought that the ability to target the fibrillar aggregates associated with exfoliation may offer a new therapeutic approach, facilitating their direct removal from affected tissues. Phage display techniques yielded two peptides (LPSYNLHPHVPP, IPLLNPGSMQLS) that could differentiate between exfoliative and non-affected regions of the human lens capsule. These peptides were conjugated to magnetic particles using click chemistry to investigate their ability in targeting and removing exfoliation materials from the anterior human lens capsule. The behavior of the fibrillar materials upon binding to these magnetic particles was assessed using magnetic pins and rotating magnetic fields of various strengths. Ex vivo studies showed that the magnetic particle-peptide conjugates could generate enough mechanical force to remove large aggregates of exfoliation materials from the lens capsule when exposed to a low-frequency rotating magnetic field (5000 G, 20 Hz). Biocompatibility of targeting peptides with and without conjugated magnetic particles was confirmed using MTT cell toxicity assay, live/dead cell viability assay, and DNA fragmentation studies on primary cultured human trabecular meshwork cells. This is a novel, minimally invasive, therapeutic approach for the treatment of exfoliation glaucoma via the targeting and removal of exfoliation materials that could be applied to all tissues within the anterior segment of the eye.

Structural polymorphisms in fibrillar aggregates associated with exfoliation syndrome.

Exfoliation syndrome is largely considered an age-related disease that presents with fibrillar aggregates in the front part of the eye. A growing body of literature has investigated structural diversity of amyloids and fibrillar aggregates associated with neurodegenerative disease. However, in case of exfoliation syndrome, there is a dearth of information on the biophysical characteristics of these fibrils and structural polymorphisms. Herein, structural diversity of fibrils isolated from the anterior lens capsule of patients was evaluated using transmission electron microscopy techniques. It was apparent that, despite having a low sample number of different patients, there exists a wide range of fibril morphologies. As it is not precisely understood how these fibrils form, or what they are composed of, it is difficult to postulate a mechanism responsible for these differences in fibril structure. However, it is apparent that there is a wider range of fibril structure than initially appreciated. Moreover, these data may suggest the variance in fibril structure arises from patient-specific fibril composition and/or formation mechanisms.

Recent advances in risk factors associated with ocular exfoliation syndrome.

Exfoliation syndrome is generally considered a progressive age-related systemic disorder of the extracellular matrix, which is clinically characterized through the observation of flaky white aggregates on ocular tissues. Exfoliation syndrome is directly linked to exfoliative glaucoma in elderly patients, where it is known as the most common identifiable cause of open-angle glaucoma. Despite the identification of various risk factors associated with exfoliation syndrome, the exact pathogenesis of this syndrome has not been fully elucidated. There is a growing number of genome-wide association studies in different populations around the world to identify genetic factors underlying exfoliation syndrome. Besides variants in LOXL1 and CACNA1A genes, new loci have been recently identified which are believed to be associated with exfoliation syndrome. Among different genetic factors, functional variants might help to better understand mechanisms underlying this systemic disorder. Besides genetic factors, epigenetic regulation of different gene expression patterns has been thought to play a role in its pathogenesis. Other factors have been also considered to be involved in the development of exfoliation syndrome at cellular organelles level where mitochondrial impairment and autophagy dysfunction have been suggested in relation to exfoliation syndrome. This review addresses the most recent findings on genetic factors as well as cellular and molecular mechanisms involved in both the development and progression of exfoliation syndrome.

Peptides for targeting ßB2-crystallin fibrils.

Crystallins are a major family of proteins located within the lens of the eye. Cataracts are thought to be due to the formation of insoluble fibrillar aggregates, which are largely composed of proteins from the crystallin family. Today the only cataract treatment that exists is surgery and this can be difficult to access for individuals in the developing world. Development of novel pharmacotherapeutic approaches for the treatment of cataract rests on the specific targeting of these structures. ßB2-crystallin, a member of ß-crystallin family, is a large component of the crystallin proteins within the lens, and as such was used to form model fibrils in vitro. Peptides were identified, using phage display techniques, that bound to these fibrils with high affinity. Fibrillation of recombinantly expressed human ßB2-crystallin was performed in 10% (v/v) trifluoroethanol (TFE) solution (pH 2.0) at various temperatures, and its amyloid-like structure was confirmed using Thioflavin-T (ThT) assay, transmission electron microscopy (TEM), and X-ray fiber diffraction (XRFD) analysis. Affinity of identified phage-displayed peptides were analyzed using enzyme-linked immunosorbent assay (ELISA). Specific binding of a cyclic peptide (CKQFKDTTC) showed the highest affinity, which was confirmed using a competitive inhibition assay.

Antiproliferative effect of H2O2 against human acute myelogenous leukemia KG1 cell line.

It has clearly been established that oxidative stress leads to perturbation of various cellular processes resulting in either inhibition of cell proliferation or cell death. In addition, there is a growing body of evidence indicating that reactive oxygen species (ROS) are required as signal molecules that regulate different physiological processes including survival or death. Free radicals, particularly ROS, have been proposed as general mediators for apoptosis and recent studies have established that the mode of cell death depends on the severity of the oxidative damage. In this study, we determined the effect of oxidative stress on cell proliferation and characterization of cell death in human KG1 cells treated with H2O2. Our results indicated that oxidative stress leads to a significant decrease in cell proliferation and induction of apoptosis. Moreover, our study suggests that antiproliferative and apoptotic cell death effects of H2O2 took place via activation of caspase-3, affecting the expression of Bcl-2 and Bax (an antiapoptotic and a proapoptotic factor, respectively), and through deactivation of catalase enzyme, leading to accumulation of intracellular ROS and depletion of intracellular ATP level.