Yoshimi Kawasaki

Retinal function with lens-induced myopia compared with form-deprivation myopia in chicks

Abstract• Background: The retina is known to be involved in the development of form-deprivation myopia (FDM); however, it is not clear whether the retinal changes that lead to lens-induced myopia (LIM) are the same as those involved in FDM. To gain insight into the retinal mechanism(s) that cause myopia, we investigated differences in the results of electroretinography (ERG) in eyes with FDM and LIM. • Methods: LIM or FDM was induced in chick eyes by placing various powers of spectacles or an occluder over the left eyes of 6-day-old chicks. After 6 days, the spectacles or occluder was removed, refraction and axial length were measured and ERG was performed. Results for eyes treated with spectacles and those treated with occluders were compared.• Results: Refraction and axial length changed concomitant with the power of the lens used, but components of the ERG of eyes with LIM were not related to the power of lens added. Refraction and axial lengths of eyes covered with a — 16 D lens did not differ from these values in eyes covered with an occluder. The a- and b-waves were also similar for the two groups. However, oscillatory potentials decreased significantly in the chicks with FDM. • Conclusion: Retinal function differs in LIM and FDM, as indicated by differences in the oscillatory potentials. This difference may stem from the fact that in FDM the retinal image is continuously defocused, whereas images are ultimately focused on the retina in LIM.

GLYCATION OF APOLIPOPROTEIN E IMPAIRS ITS BINDING TO HEPARIN : IDENTIFICATION OF THE MAJOR GLYCATION SITE

The increased glycation of plasma apolipoproteins represents a possible major factor for lipid disturbances and accelerated atherogenesis in diabetic patients. The glycation of apolipoprotein E (apoE), a key lipid-transport protein in plasma, was studied both in vivo and in vitro. ApoE was shown to be glycated in plasma very low density lipoproteins of both normal subjects and hyperglycemic, diabetic patients. However, diabetic patients with hyperglycemia showed a 2-3-fold increased level of apoE glycation. ApoE from diabetic plasma showed decreased binding to heparin compared to normal plasma apoE. The rate of Amadori product formation in apoE in vitro was similar to that for albumin and apolipoproteins A-I and A-II. The glycation of apoE in vitro significantly decreased its ability to bind to heparin, a critical process in the sequestration and uptake of apoE-containing lipoproteins by cells. Diethylenetriaminepentaacetic acid, a transition metal chelator, had no effect on the loss of apoE heparin-binding activity, suggesting that glycation rather than glycoxidation is responsible for this effect. In contrast, glycation had no effect on the interaction of apoE with amyloid beta-peptide. ApoE glycation was demonstrated to be isoform-specific. ApoE(2) showed a higher glycation rate and the following order was observed: apoE(2)>apoE(4)>apoE(3). The major glycated site of apoE was found to be Lys-75. These findings suggest that apoE is glycated in an isoform-specific manner and that the glycation, in turn, significantly decreases apoE heparin-binding activity. We propose that apoE glycation impairs lipoprotein-cell interactions, which are mediated via heparan sulfate proteoglycans and may result in the enhancement of lipid abnormalities in hyperglycemic, diabetic patients.

Specific detections of the early process of the glycation reaction by fructose and glucose in diabetic rat lens

The glycation reaction by fructose, as well as that by glucose, in control and diabetic rat lens was analyzed by using antibodies which specifically recognize adducts of lysine with fructose and with glucose. Levels of fructose adducts in diabetic rat lens were 2.5 times that of the control, and correlated with sorbitol levels. This was mainly due to enhanced glycation of β‐ and γ‐crystallins by fructose under diabetic conditions. These data suggest that glycation by fructose may also play a role in cataract formation under conditions of diabetes and aging.

Effect of a Nitric Oxide Synthase Inhibitor on Lens-Induced Myopia

Purpose: It has still not been determined whether the retinal mechanism causing form-deprivation myopia (FDM) is different from that causing lens-induced myopia (LIM). We previously reported that FDM was blocked by an intravitreal injection of the nitric oxide (NO) synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME). In this study, we investigated the effect of L-NAME on LIM in chicks. Method: The left eyes of 6-day-old chicks were injected with 30 µl of nontoxic concentrations of L-NAME (≤360 mM) or saline. The right eyes were injected with 30 µl of saline. A –16 dpt lens was placed in front of the left eye for 6 days. Another group of 6 chicks were injected with 180 mML-NAME (left eye) and with saline (right eye) before placing –16 dpt lenses in front of both eyes. After removing the lens, the refraction and the axial length were measured. The effect of L-NAME (180 mM) on the retina of a separate group of chicks was examined by electroretinography 60 min after an intravitreal injection in non-LIM-treated eyes. Results: The eyes of chicks that were injected with 180 or 360 mML-NAME were less myopic and had significantly shorter axial lengths than control eyes. A significant decrease of the On response and an increase of the Off response were observed. Conclusion: The injection of L-NAME into developing chick eyes that were then covered with a –16 dpt lens resulted in a modifications of retinal function and an inhibition of the development of myopia. These results, combined with the earlier findings, suggest that NO modulates a common retinal pathway that leads to both LIM and FDM.

Retinal Glial Cells Stimulate Microvascular Pericyte Proliferation Via Fibroblast Growth Factor and Platelet-derived Growth Factor In Vitro

PURPOSE To investigate whether retinal glial cells (RGCs), which are believed to play an important role in the development and maintenance of microvessels, stimulate the proliferation of retinal bovine microvascular pericytes, an essential component of the vessels. METHODS Conditioned medium (CM) was collected from a primary culture of RGC obtained from chick embryonic retina. The cell number was assayed after stimulation by RGC-CM. Also, by neutralizing antibody and reverse transcription polymerase chain reaction (RT-PCR), we tried to identify which factor of the RGCs contributes to the pericyte stimulation. RESULTS Pericyte proliferation was stimulated by RGC-CM in a dose-dependent manner. Platelet-derived growth factor-BB (PDGF-BB), acidic fibroblast growth factor (aFGF), and basic fibroblast growth factor (bFGF) stimulated pericyte proliferation; however, PDGF-AA, transforming growth factor-beta2 (TGF-beta2), and vascular endothelial growth factor (VEGF) did not. The RGC-CM-dependent stimulative effect was blocked, in part, by the neutralizing antibodies for aFGF, bFGF, and PDGF. A mixture of these three antibodies completely blocked the stimulation. RT-PCR revealed that RNA for aFGF, bFGF, and TGF-beta2 were expressed in RGCs. CONCLUSIONS Pericyte growth is stimulated in vitro by RGC-CM through aFGF, bFGF, PDGF-BB, at least in part. This finding suggests that RGCs may modulate in vivo pericyte cell growth through these three growth factors.

The Effect of Nitric Oxide on the Contractile Tone of Müller Cells

The effect of nitric oxide (NO) on the contractile tone of Müller cells was investigated. Müller cells, isolated from the retina of chick embryos, were cultured on thin sheets of silicone. S-Nitro-N-acetyl-DL-penicillamine (SNAP), an NO donor, was applied in the presence or the absence of carboxyl phenyltetramethylimidazole oxide (C-PTIO), an NO scavenger. The contractile tone of Müller cells was assessed by the extent of wrinkles created on the silicone sheets. The change of contractile tone was evaluated quantitatively by digitizing the photograph before and after the application of SNAP. Relaxation of wrinkles was induced by SNAP. C-PTIO inhibited the SNAP-induced relaxation of wrinkles. These results suggest that NO affects the contractility of embryonic Müller cells and could thus modulate ocular development.

Physiological Relevance of Aldehyde Reductase and Aldose Reductase Gene Expression

Carbonyl compounds which are produced as intermediate metabolism during ordinary metabom or are present in food or drugs are known to be toxic to living organisms because of their high degree of reactivity. It has also been suggested that elevation in protein carbonyl groups is also a likely cause of aging (Stadtman, 1992). Cells contain defense systems against these compounds (Flynn, 1982) in the form of aldo-keto reductases, which includle aldehyde and aldose reductases, which catalyze the reduction of a variety of aldehydes to alcohols in an NADPH-dependent manner (Jez et al., 1997). Cytotoxic compounds which tain an aldehyde moiety, such as tripeptidyl aldehyde (Inoue et al., 1993),trioses (Van der Jagt et al., 1992) and methotrexate (Callahan and Beverley, 1992) are deified by enzymes in this gene family. The glycation reaction represents another source carbonyl compounds. This reaction occurs during normal aging and at accelerated rates diabetes, and is involved in the pathogenesis of diabetic complications (Fujii et al. 998). Glycation alters the activity of some enzymes such as Cu,Zn-SOD (Arai et al., 1; Ookawara et al., 1992), carbonic anhydrase (Kondo et al., 1987), sorbitol dehy- drogen (Hoshi et al., 1996), and aldehyde reductase (Takahashi et al., 1995b), and is also involved in production of dicarbonyl compounds such as 3-deoxyglucosone and methyloxal (Figure 1). The cross-linking of long-lived proteins such as collagen and lens crystallins are induced by these compounds and correlates with aging and diabetes. These dicarbonyl compounds are highly toxic and induce apoptosis in susceptible cells (Okado al., 1996). The production of aldehydes is enhanced during pathological conditions, includ diabetes and cancer. Concomitantly, aldehyde-reducing activity is also increased in hepatoma cell lines (Canuto et al., 1994). This is mainly due to an elevated expresn of the aldose reductase gene (AKR1B) as has been demonstrated in chemically induceepatoma tissues (Zeindl-Eberhart et al., 1994; Takahashi et al., 1995a).

Reducing Sugars Induce Apoptosis in Pancreatic β-Cells by Provoking Oxidative Stress via a Glycation Reaction

Reducing sugars brought about apoptosis in isolated rat pancreatic islet cells as well as in a pancreatic β-cell-derived cell line, HIT. This apoptosis was characterized biochemically by internucleosomal DNA cleavage and morphologically by nuclear shrinkage, chromatic condensation, and apoptotic body formation. N-acetyl-L-cysteine and aminoguanidine inhibited the apoptosis. Proteins in β-cells were actually glycated by the binding with an antibody that can specifically recognize the protein glycated by fructose. The FACS analysis using dichlorofluorescin diacetate showed that reducing sugars increased intracellular peroxide levels preceding the induction of apoptosis. Levels of carbonyl and malondialdehyde were also increased. These results suggest that reducing sugars trigger oxidative modification and apoptosis in pancreatic β-cells by provoking oxidative stress mainly via a glycation reaction, which may explain the deterioration of β-cells under diabetic conditions.

ERG of Lens-Induced Versus Form-Deprivation Myopia in Chicks

The retina is known to be involved in the development of form-deprivation myopia (FDM); however, it is not clear if the retinal changes that lead to lens-induced myopia (LIM) are the same as those involved in FDM. To gain insight into the retinal mechanism(s) that cause myopia, we investigated differences in the results of electroretinography (ERG) in eyes with FDM and LIM. LIM or FDM was induced in chick eyes by placing various powers of spectacles or an occluder over the left eyes of 6-day-old chicks. After 6 days, the spectacles or occluder was removed, refraction was measured, and ERG was performed. Results for eyes treated with spectacles and those treated with occluders were compared. Refraction changed concomitant with the power of the lens used. Refraction and axial lengths of eyes covered with a–16 D lens did not differ from these values in eyes covered with an occluder. The a- and b-waves were also similar for the two groups. However, oscillatory potentials decreased significantly in the chicks with FDM. We concluded that retinal function differs in LIM and FDM, as indicated by differences in the oscillatory potentials. This difference may stem from the fact that in FDM the retinal image is continuously defocused, whereas images are ultimately focused on the retina in LIM.