on line edition The role of lens epithelial cells in PCO and lens regeneration Pr OfessiOnal ar TiCle id The role of lens epithelial cells in PCO and lens regeneration Department of Ophthalmology, University Medical Centre Ljubljana, Ljubljana Correspondence: sofija andjelic, e: sofija.andjelic@kclj.si Key words: lens epithelial cells; lens capsular bag; cell pluripotency; cell proliferation; migration of cells Cite as: Zdrav Vestn. 2017; 86:236–43. r eceived: 16. 11. 2016 accepted: 6. 4. 2017 Zdrav Vestn | May – June 2017 | Volume 86 neurobiology Professional article The role of lens epithelial cells in the development of the posterior capsule opacification and in the lens regeneration after congenital cataract surgery sofija andjelic Abstract Posterior capsule opacification–PCO is the most common complication after cataract surgery. Pro- liferation and migration of lens epithelial cells that remain in the capsular bag following cataract surgery can lead to the development of PCO, which is the main cause of deterioration of visual func- tion. PCO shows the classic features of fibrosis, including hyperproliferation, migration, deposition of matrix and its shrinkage and transdifferentiation into myofibroblast. Astonishingly, the results of recent research show the importance of lens epithelium for lens re- generation following congenital cataract surgery. New minimally invasive cataract surgery removes only 1–1.5 mm of lens epithelium more laterally, so the major part of the epithelium remains in the capsular bag. Conceptually, the new method differs from the current practice, since it preserves the endogenous epithelial cells of the lens and achieves functional lens regeneration in rabbits and mon- keys as well as in human infants with congenital cataracts. Pluripotency of lens epithelial cells and their stem cell nature are crucial for lens regeneration. Ex vivo cultures of the lens capsule explants can be used for testing the pharmacological agents for stimulating and inhibiting the growth of lens epithelial cells. Functionality of the cells and responses to pharmacological agents can be studied by analysing the intra- and inter-cellular calcium (Ca 2+ ) signalling. Stimulating the growth of lens epithelial cells is important in lens regeneration while in- hibiting the growth of lens epithelial cells is important in preventing the development of PCO. In the article I described the methods for the analysis of lens epithelial cells after cataract surgeries, which are carried out in the laboratory of the Eye Hospital, University Medical Centre Ljubljana. Cite as: Zdrav Vestn. 2017; 86:236–43. 1. Introduction Cataracts are the most common cause of vision impairment in humans. One talks about cataract, when the transpar- ency of the lens is reduced, which pre- vents the light from smooth transition to the back of the eye, thereby worsening vision. According to the World Health Organization, it is estimated that about 20 million people today have bilateral visual impairment due to cataract, this number is expected to reach 50 million by 2050  (1). Currently, the main method of treatment is surgical, so the cataract surgery is the most common eye surgery. on line edition Zdrav Vestn | May – June 2017 | Volume 86 neur ObiOl Ogy The lens is composed of three parts: the capsule, the cortex and the nucleus (Figure 1). The lens capsule is a thin transparent membrane that wraps the lens. It is composed mainly of collagen type IV and laminin  (2). The cortex consists of epithelial cells, which gradu- ally turn into fibres. A single-layered lens epithelium is located anteriorly, under the lens capsule. Lens epithelium cells have their apical surface facing inwards towards the nucleus and the basal sur- face outward, touching the lens capsule. The lens epithelium is the first physical and biological barrier of the lens and the metabolically the most active part, maintaining the physiological balance. The ionic pump in the lens epithelial cells maintains homeostasis and clarity of the lens. The anterior lens epithelium also serves as a source of precursor cells, from which the fibre cells are formed by molecular and morphological differen- tiation. The lens nucleus is composed of the oldest cells, virtually without metabol- ic activity  (3). It consists of a mass of compressed fibre cells that are covered anteriorly with single-layer epithelium and wrapped with the lens capsule. The amount of the fibre cells provides the functional properties of the lens. 2. The posterior capsule opacification (PCO) development In cataract surgery (phacoemulsifica- tion), after removal of the anterior lens capsule together with lens epithelial cells measuring approximately 5.5 mm, the cortex and the nucleus of the natural eye lens are removed and a new artifi- cial lens is inserted. The surgery is very safe and rarely associated with complica- tions, however, relatively often posterior capsule opacification (PCO) occurs after the surgery. Upon the cataract surgery, the attenuation of visual acuity due to the PCO may develop in approximate- ly 20 % of cases within a period of five years  (4). PCO usually develops from the lens epithelial cells that remain on the lens capsule after cataract surgery (phacoemuslification)  (4,5). During the PCO formation, rather than the poste- rior lens capsule itself becoming opaque, opaque secondary membranes develop on the posterior lens capsule (6-8). The secondary membranes are formed by proliferation, migration  (9) and trans - formation (so-called epithelial-mesen- chymal transition) of the lens epithelial cells (10,11). Thus modified cells are ca- pable of depositing the collagen and re- storing or regenerating lens fibres in the area between the artificial intraocular lens and the posterior lens capsule (12). Lens epithelial cells proliferate in various patterns. Clinically, two basic morpho- logical forms of PCO are distinguished: fibrous and pearl. The first is formed by the proliferation and migration of the lens epithelial cells, which are located under the anterior lens capsule and the passage in myofibroblast (so-called ep- ithelial-mesenchymal transition)  (13). The second form, with the pearls, is formed from the lens epithelial cells, The role of lens epithelial cells in PCO and lens regeneration Figure 1: l ens structure schematic presentation. on line edition The role of lens epithelial cells in PCO and lens regeneration Pr OfessiOnal ar TiCle which are located in the equatorial part of the lens or the lens pole and regen- erate into the lens fibres, which express crystalline (5,14). 3. Lens regeneration or renewal The latest research results show that the lens can renew–regenerate after a new way of congenital cataracts surgery, where the key is the lens epithelium. The new way of congenital cataracts surgery preserves larger part of lens epithelial cells, which then proliferate and form a functional lens. The surgery turned out to be successful in both rabbits and monkeys, as well as in children with con- genital cataracts (15). Cataract is one of the major causes of visual impairment in children (16). The present method of cataract surgery in children includes the creation of a large opening in the anterior lens capsule with a diameter of 6 mm, resulting in a large area of the removed capsule, and thus the removal of a large number of lens ep- ithelial cells. In this way, the incidence of PCO is reduced. Recently, a new way of congenital cataract surgery has been de- scribed, in which the aperture of a small- er diameter is formed on the periphery of the anterior lens capsule (<1.5 mm) in order to enable lens regeneration. This leaves more lens epithelial cells in the lens capsular bag, which give rise to a new functional lens. Twelve children with congenital cata- ract (24 eyes) had a new minimally in- vasive surgery which allows lens regen- eration. The control group of 25 children with congenital cataract (50 eyes) had a standard cataract surgery  (15). The new method of surgery has two advantages: it considerably reduces the size of the removed lens capsule and at the same time moves the opening of the lens cap- sule from the central visual axis to the periphery. After the surgery, the cornea, anterior chamber and fundus were clear. Complications associated with surgery have not been detected. Openings in the anterior lens capsule healed in one month. Renewed clear biconvex lens structure was formed three months after the surgery. After six months, no compli- cations or unorganized tissue regenera- tion were recorded. The new minimally invasive surgical method provides clear visual axis while preserving lens epithelial cells that have regenerative potential. Lens epithelial cells in the adult hu- man eyes show an increased regenera- tive potential after injury (15), which in- dicates the possibility of functional lense renewal also in older patients with age- related cataracts. 4. Preventing or promoting the growth of lens epithelial cells In the new mode of congenital cata- ract surgery, lens epithelial cells are re- sponsible for both PCO as well as the emergence of a new lens. Study of the inhibitory mechanisms of the epithelial transition to mesen- chyme is important to prevent the devel- opment of PCO. It has been demonstrat- ed that transforming growth factor  β, TGFβ, induces a transition of epithelium to mesenchyme, which is central in the formation of fibrous PCO, probably via the ERK / MAPK signalling pathway (chain of proteins in the cell that trans- mits a signal from the cell surface recep- tors to the DNA in the cell nucleus) (17). Fibronectin type III in the extracellular matrix protein tenascin-C plays a role in inhibiting the epithelial-mesenchymal transition (18). On the other hand, the study of mechanisms of stimulating the differ- The role of lens epithelial cells in PCO and lens regeneration on line edition Zdrav Vestn | May – June 2017 | Volume 86 neur ObiOl Ogy The role of lens epithelial cells in PCO and lens regeneration entiation of lens epithelial cells to fibre cells is important for the reconstruction of functional lens. Growth factors that act through tyrosine kinase receptors (RTKs), such as fibroblast growth factors (FGF), have normal regulatory role in the lens (19). Today it is known that the process of differentiation of lens epithe- lial cells to fibre cells is driven by FGF. Little is known about the factors that coordinate the detailed arrangements of fibre cells in functional lens. Recent re- search gives insight into FGF- activated mechanism that involves the interaction of Wnt-Frizzled and Jagged / Notch sig- nalling pathways. Wnt and Notch signal- ling pathway are used by animal cells to control their identity and behaviour during development. Mutual interaction of lens epithelial cells and fibre cells is crucial for merging and maintaining a regulated three-dimensional structure, which is central to the functioning of the lens (17). Study of lens epithelial cells prolif- eration is important as regarding the development of PCO the proliferation should be prevented, while regarding the formation of a new lens the proliferation should be stimulated. The proliferation grade of lens epithelial cells is age-de- pendent. In humans, lens epithelial cells have decreased responsiveness to mito- genic growth factor, FGF-2  (20). This is consistent with the drop in the density of human lens epithelial cells in the central area of the lens epithelium with age (21). Ki-67 nuclear protein is a cell marker associated with cell proliferation. Some of the Ki-67-positive cells were also de- tected in the central, anterior region of the epithelium in young bovine and hu- man lens, where a younger lens has more proliferating cells than an older one (22). Lens epithelial cells proliferation was studied in the adult mice lens epithelium, where they found two groups of cells: slower, rarely cycling cells, which are lo- cated in the central area of lens epitheli- um, and more frequently cycling cells in the peripheral or germinative area (23). There is a relatively low percentage of the first cells (2–3 %), so the authors sug- gest that these are lens epithelial stem cells, which rarely divide during homeo- stasis. With the disturbance, these cells start proliferating and provide the divi- sion, which will supply the central and germinate area with the cells that have the capability of further division. The interaction of the apical surfaces of lens epithelial cells, which are facing inwardly towards the nucleus, with the inner por- tion of the lens, where they contact the apical surfaces of the fibre cells, regulates Figure 2: an example of an attached human anterior lens capsule explant with growing lens epithelial cells in a Petri dish. in the bottom panels, two areas of the lens capsule with growing epithelial cells are shown at larger magnification. on line edition The role of lens epithelial cells in PCO and lens regeneration Pr OfessiOnal ar TiCle lens epithelial cells proliferation (24,25). BMI-1, a protein which in humans is en- coded by the BMI-1 gene (B cell-specific murine leukemia virus), is necessary for the maintenance and renewal of the en- dogenous lens epithelial cells. Loss of the BMI-1 leads to a decrease in the prolifer - ative capacity of lens epithelial cells and the formation of cataracts (15). 5. The methods of analysis of lens epithelial cells after cataract surgery at our laboratory To study the role of the human lens epithelium, both its regenerative poten- tial and the PCO development, the an- terior lens capsule obtained from cata- ract surgery can be used  (26), to grow ex vivo explant cultures of the lens cap- sule (27,28). At the Eye Clinic Ljubljana we use anterior lens capsules obtained via standard cataract surgery, which are normally discarded. The lens epithelium structure and differences of apical and basal side we have shown using scan- ning and transmission electron micros- copy and confocal microscope. With each of the three methods used we have shown the same morphological features, the extensions and the entanglements of the cytoplasmic membrane of lens epi- thelial cells at the border with the lens capsule, while it has been shown that the apical surface of lens epithelial cells is smooth (29). Using scanning and trans- mission electron microscopy, we stud- ied the lens epithelium in patients with different cataract types. In patients with intumescent cataracts the lens epithe- lium has the following features: swollen cells, spherical formations and degraded cells  (30). In patients with retinitis pig- mentosa we observed the following features of the lens epithelium, such as holes and the degradation of the epi- thelium measuring from <1 μm to more than 50 μm  (31). The holes in the lens ep- ithelium likely have a role in the cataract development. Homeostasis of the intra- cellular calcium (Ca 2+ ) concentration is a general indicator of the functioning of cells. In the study of the role of the lens epithelium and the analysis of the in- tra- and intercellular Ca 2+ signaling in human anterior lens epithelial cells we showed that in more developed cataracts, cells were found to exhibit a slower col- lective response to stimulation and a less pronounced spatio-temporal clustering of cells. The intercellular networks were found to be sparser and more segregated than in mild cataracts (32). We have also shown the contraction of lens epithe- lial cells upon nonspecific stimulation, The role of lens epithelial cells in PCO and lens regeneration Figure 3: an example of changes in the intracellular calcium concentration of the lens epithelial cells grown in culture of lens capsule explant, in response to mechanical stimulation. The stimulation site is indicated by an arrow in the first picture, which shows the cell morphology at 360 nm. Time changes in intracellular calcium concentrations for 5 selected areas are shown below; the areas are marked with circles of the same colour. Colour images through 360/380 nm show the rise in calcium concentration at selected moments: red colour shows the highest concentration, yellow smaller and green the smallest. on line edition Zdrav Vestn | May – June 2017 | Volume 86 neur ObiOl Ogy The role of lens epithelial cells in PCO and lens regeneration which is at least partly independent of the changes in intracellular Ca 2+ concen- tration. Contraction can be induced by a mechanical stimulus, the response is fast and after the cessation of stimulation, cells tend to return to the initial non- contracted state  (33). This contraction of cells can lead to higher water perme- ability, which could be the mechanism of the formation of cataracts upon the in- sertion of the phakic lens, if the latter are touching the anterior lens capsule. In connection with the development of PCO and lens regeneration, we have shown that the anterior lens epithelium of an adult has pluripotent lens epithe- lial cells. Cultured human anterior lens capsule harbours lens epithelial cells that can proliferate and migrate, suggesting their pluripotency or putative stem cell nature. By immunostaining, we have shown that the Ki-67 nuclear protein that is associated with cell proliferation, and Sox2 transcription factor that is es- sential for pluripotency of undifferenti- ated embryonic stem cells, are expressed in the lens epithelial cells grown on the human anterior lens capsule  (27). Stem and progenitor cells expressing Sox2 are important for tissue regeneration and survival in mice (34). Anterior lens capsules obtained via cataract surgery we also use for the cul- tivation of ex vivo explants of the lens capsule by plating the lens capsule in a Petri dish. For lens epithelial cells to mi- grate to the glass Petri dishes, the lens capsule must be attached to the bottom of the Petri dish. We have developed a method for attaching the explants using viscoelastic  (28). An example of the at- tached human anterior lens capsule ex- plant with growing lens epithelial cells is shown in Figure 2. Ex-vivo cultured ex- plant of the lens capsule with the growing lens epithelial cells can serve as a model for testing a variety of physical and phar- macological effects. We have shown that targeted and localized microplasma causes dose-dependent morphological changes of cells and apoptosis of ex vivo cultured human lens epithelial cells. The results show that the single cell specific micropipette plasma can be used to se- lectively induce death in lens epithelial cells which remain in the capsular bag after cataract surgery and thus prevent their migration to the posterior lens cap- sule and PCO formation (35). The func- tion of the growing lens epithelial cells of lens capsule explants can be analyzed by means of measurements which show changes in the intracellular Ca 2+ concen- tration, which is an indicator of the cell functioning, as a response to a variety of physical and pharmacological stimuli. Figure 3 shows an example of changes in the intracellular Ca 2+ concentration of the lens epithelial cells grown in culture from lens capsule explant, in response to mechanical stimulation, and shows in- tercellular Ca 2+ signalling. The raise in Ca 2+ concentration is not simultaneous in all the selected areas but shows a time delay, which indicates that the signal travels. The intercellular Ca 2+ signalling indicates functional contacts between the cells that can be compared between the confluent and non-confluent lens epithelial cells. Similar studies are im- portant for testing the pharmacological agents and studying the promotion and inhibition of lens epithelial cell growth. 6. Conclusions The lens epithelial cells are respon- sible for both PCO as well as the lens re- generation after the new way of congeni- tal cataract surgery. The new minimally invasive method of congenital cataract surgery in children preserves endog- enous stem lens epithelial cells, thereby achieving functional lens regeneration. on line edition The role of lens epithelial cells in PCO and lens regeneration Pr OfessiOnal ar TiCle The pluripotency of lens epithelial cells, which have the stem cell properties, is of key importance for the regeneration of the lens. Ex vivo cultures of human lens epithelial cells can serve as a model for testing various physical and pharmaco- logical effects for promoting or inhibit- ing the cell growth. The functionality of cells and the responses to various stimu- lations can be studied by analysing the intra- and intercellular Ca 2+ signalling. 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