Kiyoyuki Majima

Concentration of Transforming Growth Factor β2 in Aqueous Humor

The total transforming growth factor (TGF) β2 concentration in the anterior chamber aqueous humor of 96 cataract patients with ages ranging from 17 to 88 years was measured using ELISA to investigate the changes that occur with age, difference of axial length, difference of localization of opacification of the cataractous lens and complications with other eye diseases. It was found that the total TGF-β2 concentration (1) decreases with age, (2) shows slight changes with axial length, (3) has slight changes with difference of localization of opacification, (4) is significantly high in patients with concurrent open-angle glaucoma (p < 0.05), (5) is high in patients with complicating diabetes who have undergone panretinal photocoagulation for diabetic retinopathy (p < 0.05) and (6) is low in patients with atopic cataracts. There have been several reports on point 4 above, but none to date of the other points. These findings provide useful information on the intraocular activity of TGF-β2.

A study of the proliferating activity in lens epithelium and the identification of tissue-type stem cells

Using a newly established fixation method and immunohistochemical methods, we precisely described the regions of cells stained with various antibodies relating to cell proliferation; this method enabled us to make cellular-level diagrams of the epithelium in which the position of every lens epithelial cell (LEC) was determined in reference to the cell located at the top of the bow area. The proliferating activity of LECs of 4-week-old (4W) mice was examined either by labeling with 5-bromodeoxyuridine (BrdU) in vivo or by measuring the amount of mRNA prepared from LECs, which had been separated into the posterior part, containing the germinative zone, and the anterior part and then cultured. The epithelial region stained with antibody for proliferating cell nuclear antigen (PCNA) and cyclin D1 remained relatively constant during the study period, although the positive region was reduced a little from embryonic day 18 (E18) to 12W. This region at 4W overlapped well with the DNA synthesizing region. Therefore, we reasoned that this region would correspond to the germinative zone of the adult mouse. Considering together with results of the reactivation pattern of genes, we considered that the location of tissue-type stem cells in lens epithelium (LE) as immediately anterior to the germinative zone.

Human Lens Epithelial Cells Proliferate in Response to Exogenous EGF and Have EGF and EGF Receptor

While epidermal growth factor (EGF) has been found to exist in the human aqueous humor, which is necessary for maintaining the physiological condition of the lens, our immunohistochemical study confirmed the presence of EGF receptors and EGF in human lens epithelial cells (LECs), with EGF promoting growth potential and morphological change of the cells. Based on these findings, we feel that the effect of EGF upon human LECs is related to the movement of the cells in vivo.

The relationship between morphological changes of lens epithelial cells and intraocular lens optic material

To examine the morphological changes of lens epithelial cells (LECs) occurring directly beneath and at regions contacting various intraocular lens (IOL) optic materials, human LECs were cultured on human anterior lens capsules and were further incubated upon placing above the cells lens optics made of polymethylmethacrylate, silicone, and soft acrylic material. Observations as to the morphological changes of LECs under phase-contrast microscope and scanning electron microscope were performed on the 14th day of incubation. Gatherings of LECs were observed at regions contacting the soft acrylic material under phase-contrast microscope, and gatherings of LECs were observed accurately at the same regions mentioned above under scanning electron microscope. On the other hand, LECs in contact with two other optic materials did not show morphological changes. The results suggest that LECs attached to and proliferated on not only the anterior lens capsules but also the soft acrylic IOL optics. The model used in this study may be useful in studying the relationship between cellular movement of LECs and IOL optic material.

Histopathological and cell biological analyses of the formation mechanism of anterior polar cataract.

Anterior polar cataract, defined as an opacification limited to the anterior polar region of the crystalline lens, results in a significant decline of vision even though the area of opacification is generally small. Having performed histopathological and cell biological studies in an attempt to identify the formation mechanism of anterior polar cataract, we found that lens epithelial cells (LECs) in contact with the opacified region were arranged in a monolayer and contained vacuoles. Furthermore, compared to LECs obtained from cortical and nuclear cataractous lenses and cultured for the study, these LECs revealed a significantly diminished proliferation, suggesting that their physiological activity had been reduced. On the other hand, LECs existing within the opacified region were multi-layered, and there were structureless deposits including remnants of organelles among the cells. Based on these findings, we have concluded that the formation mechanism of anterior polar cataract is as follows: LECs contacting the opacified region initially undergo significant mitosis; rather than migrating towards the equator, these proliferating cells accumulate and form a multilayer in the anterior polar region. Since multi-layered cells are surrounded by the anterior lens capsule and the lens cortex, there is a limit as to how many layers the cells can continue to form; when this limit is reached, these cells next undergo endocytosis and exocytosis. Because the physiological activity has been reduced as a result of having undergone significant proliferation, the cells contacting the opacified area are no longer able to force the proliferating cells towards the anterior polar region; hence multi-layering of the cells and the entire cellular movement are terminated at this point and result in cells with vacuoles and diminished physiological activity. While further studies are required to confirm our hypothesis, we believe this is the first study to suggest a possible mechanism of formation of anterior polar cataract.

Effect of Epidermal Growth Factor upon Morphological Changes of Human Lens Epithelial Cells

Phase-contrast and transmission electron microscopic studies were performed to evaluate the effect of epidermal growth factor (EGF) upon the morphology of lens epithelial cells (LECs) obtained from human cataractous lenses, cultured on human anterior lens capsules, and divided into the following three groups: group 1 receiving no EGF supplement, group 2 supplemented with 1 ng/ml EGF, and group 3 supplemented with 10 ng/ml EGF. Phase-contrast microscopy revealed that the cells in the group supplemented with 1 ng/ml EGF concentration had elogated, and those supplemented with 10 ng/ml EGF disclosed changes indicating fibroblast-like cells. Under transmission electron microscopy, the cells in the group supplemented with 1 ng/ml EGF concentration had become multilayered and a few cells had a ball-and-socket junction structure and nucleolar chromatin condensation, while those supplemented with 10 ng/ml EGF showed changes not only in the cells but also in the anterior lens capsules to present numerous microfibers. This is the first study to confirm the effect of EGF upon LECs cultured on anterior lens capsules, a condition closely resembling the intraocular environment, and these findings are significant.

Presence of Growth Factor in Human Vitreous

The aqueous humor and the vitreous not only maintain integrity of the eye but also play a vital role in providing nutrients to ocular tissues. While the presence of epidermal growth factor, basic fibroblast growth factor, and transforming growth factor-beta (TGF-beta) have been confirmed in human aqueous humor, only TGF-beta has been found to exist in human vitreous. In this study I obtained eyes for keratoplasty from 20 male and 20 female donors, or a total of 40 subjects; upon removing the corneal button. I extracted 500 microliters of the vitreous humor in order to confirm, using the ELISA method, the presence of transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF) in the vitreous. TGF-alpha ranging from 78 to 250 pg/ml, or an average of 130.65 +/- 40.89 pg/ml, was found in all eyes. On the other hand, EGF was not confirmed in all eyes. This is the first study to confirm the presence of TGF-alpha in the human vitreous.

Cell biological analysis of the human cataractous lens: implication of lens epithelial cells in the development of aftercataract.

Lens epithelial cells (LECs) from 3 patients with congenital cataract aged 2, 4 and 12 months, respectively, were cultured. The findings were compared with those from 26 eyes of 26 patients with senile cataract, with ages ranging from 60 to 89 years. The significantly higher growth potential of LECs in patients with congenital cataract compared to those with senile cataract, seems to be the cause of the higher frequency of aftercataract following cataract surgery in infants.

Cell Biological Analysis with Respect to Cause of Fibrous Opacification of the Anterior Capsule after Cataract Extraction

It has been assumed that lens epithelial cells (LECs) existing at the capsulotomy edge have been traumatized through anterior capsulotomy in cataract extraction. In this study, the correlation between traumatized LECs remaining at the anterior capsulotomy edge and epidermal growth factor (EGF) found in the aqueous humor, a cell growth factor though to affect cell morphology, was determined. Anterior lens capsules with adhering LECs were obtained following anterior capsulotomy performed during cataract surgery to first confirm the presence of EGF receptors on LECs, which are needed for EGF to be biologically active. Besides, to identify any EGF receptors on traumatized LECs, I next intentionally traumatized the cells by pressing them with a forceps from the anterior capsular side. It has been found that the LECs containing EGF receptors were always those existing at the edge of the anterior capsular opening and LECs containing EGF receptors existed along the pressed region too. The present results indicate that traumatized LECs along the capsulotomy edge have undergone changes to manifest EGF receptors, thus allowing EGF from the aqueous humor to become more active. The physiological effect of EGF upon these LECs may therefore be one of the causative factors of fibrous opacification of the anterior capsulotomy edge after cataract extraction.

A case of true exfoliation.

Using light and transmission electron microscopy, we have performed a histopathological evaluation of the anterior lens capsule, isolated during cataract extraction from an eye revealing a cellophane-like membrane on the lens surface of the pupil area. In addition to light microscopy showing one third of the anterior capsule had partially detached, transmission electron microscopy demonstrated irregular alignment of capsular structure near the detached regions. These microscopic features, together with clinical findings, led us to believe that this is a case of true exfoliation.