miR-378a-5p and miR-630 induce lens epithelial cell apoptosis in cataract via suppression of E2F3

Cataract, an eye disease that threatens the health of millions of people, brings about severe economic burden for patients and society. MicroRNA (miR)-378a-5p and miR-630 were recognized as essential regulators in multiple cancers. However, the exact functions of miR-378a-5p and miR-630 in cataract are still unclear. The expression of miR-378a-5p, miR-630, and E2F transcription factor 3 (E2F3) in tissues and cells was measured by quantitative real-time polymerase chain reaction. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay was used to evaluate cell viability. Flow cytometry was conducted to analyze cell apoptosis. The interaction between E2F3 and miR-378a-5p or miR-630 was confirmed by dual-luciferase reporter assay. The expression of proteins E2F3, B cell lymphoma (Bcl-2), Bcl-2 associated X (Bax), and cleaved caspase 3 was detected by western blot assay. The expression of miR-378a-5p and miR-630 was up-regulated whereas E2F3 was down-regulated in human cataract lens tissues compared with normal lens tissues. Depletion of miR-378a-5p or miR-630 enhanced proliferation and reduced apoptosis of human lens epithelial cells. Interestingly, up-regulation of E2F3 exhibited the same trend. Next, dual-luciferase reporter assay validated the interaction between E2F3 and miR-378a-5p or miR-630. The rescue experiments further revealed that E2F3 knockdown could recover miR-378a-5p, and miR-630 inhibitor induced promotion of cell proliferation and inhibition of apoptosis in cataract. miR-378a-5p and miR-630 repressed proliferation and induced apoptosis of lens epithelial cells by targeting E2F3 in cataract, representing a prospective alternative therapy for cataract.

Oxidative stress of crystalline lens in rat menopausal model / O estresse oxidativo do cristalino em modelo de rata na menopausa

ABSTRACT Purpose: To evaluate lenticular oxidative stress in rat menopausal models. Methods: Forty Wistar female albino rats were included in this study. A total of thirty rats underwent oophorectomy to generate a menopausal model. Ten rats that did not undergo oophorectomy formed the control group (Group 1). From the rats that underwent oophorectomy, 10 formed the menopause control group (Group 2), 10 were administered a daily injection of methylprednisolone until the end of the study (Group 3), and the remaining 10 rats were administered intraperitoneal streptozocin to induce diabetes mellitus (Group 4). Total oxidant status (TOS), total antioxidant capacity (TAC), and oxidative stress index (OSI) measurements of the crystalline lenses were analyzed. Results: The mean OSI was the lowest in group 1 and highest in group 4. Nevertheless, the difference between the groups was not statistically significant in terms of OSI (p >0.05). The mean TOS values were similar between the groups (p >0.05), whereas the mean TAC of group 1 was significantly higher than that of the other groups (p <0.001). Conclusions: Our results indicate that menopause may not promote cataract formation.

RESUMO Objetivo: Avaliar o estresse oxidativo lenticular em modelos de ratas na menopausa. Métodos: Quarenta ratos albinos femininos tipo Wistar foram incluídos neste estudo. Trinta ratas foram submetidas à ooforectomia para gerar o modelo de menopausa e 10 ratas formaram o grupo controle (Grupo 1). Dentre as ratas ooforectomizadas, 10 formaram o grupo controle menopausa (Grupo 2), 10 ratas receberam injeção diária de metilprednisolona até ao final do estudo (Grupo 3) e 10 ratas receberam estreptozotocina por via intraperitoneal para induzir diabetes mellitus (Grupo 4). O estado oxidante total (TOS), a capacidade total antioxidante (TAC) e as medições do índice de estresse oxidativo (OSI) dos cristalinos foram analisados. Resultados: A média de OSI foi menor no grupo 1 e maior no grupo 4. Todavia, a diferença entre os grupos não foi estatisticamente significativa (p>0,05). Os valores médios TOS foram semelhantes entre os grupos (p>0,05), enquanto a média de TAC grupo 1 foi mais elevada do que nos outros grupos ( p<0,001). Conclusões: Nossos resultados indicam que a menopausa podem não promover a formação de catarata.

Benefits of early glycemic control by insulin on sensory neuropathy and cataract in diabetic rats.

While there is an emphasis on the early glycemic control for its long-term benefits in preventing microvascular complications of diabetes, the biochemical mechanisms responsible for the long-lasting effects are not clearly understood. Therefore the impact of early insulin (EI) versus late insulin (LI) treatment on diabetic sensory neuropathy and cataract in streptozotocin-induced diabetic Wistar male rats were evaluated. EI group received insulin (2.5 IU/animal, once daily) treatment from day 1 to 90 while LI group received insulin from day 60 to 90. Early insulin treatment significantly reduced the biochemical markers like glucose, triglyceride, glycated hemoglobin, thiobarbituric acid reactive substances, advanced glycation end products and ratio of reduced glutathione and oxidized glutathione in diabetic rats. The late insulin treatment failed to resist the biochemical changes in diabetic rats. Diabetic rats developed sensory neuropathy as evidenced by mechanical and thermal hyperalgesia and showed a higher incidence and severity of cataract as revealed by slit lamp examination. Early insulin treatment protected the rats from the development of neuropathy and cataract, but late insulin administration failed to do so. The results demonstrate the benefits of early glycemic control in preventing neuropathy and cataract development in diabetic rats.

Electrolyte Imbalance in Cataract Patients.

100 cataract patients of IGGMC attending Biochemistry OPD for routine Blood sugar, were estimated for serum electrolyte level i.e. Sodium, Potassium level and compared with normal healthy age related ( 50-70 yrs) control by t test. Plasma Glucose level (to rule out Diabetes) and serum Creatinine (to rule out renal disorder) in both cases and control were also studied. Result of our study shows Elevation in serum Na level in cataract pts mean 148.52 + /-4.13 meq/lt compared to control mean 139.26 +/-3.08 meq/lt (p value 0.001) which is significantly high. Normal Serum Na level is required to maintain proper water electrolyte balance across lens membrane that in turn is also responsible for maintaining lens membrane permeability. Elevation in serum Na level in cataract pts may result into its further increase in aqueous humor of lens which may lead to osmotic imbalance across lens membrane and aggravate, progression of disease. We conclude that salt restricted Diet must be advised in Cataract patients so as to maintain normal electrolyte balance which may prevent further progression of disease.

Total antioxidant capacity in Eales’ disease, uveitis & cataract.

Background & objectives: The human system possesses antioxidants that act harmoniously to neutralize the harmful oxidants. This study was aimed to evaluate the serum total antioxidant capacity (TAC) as a single parameter in Eales’ disease (ED) and in an acute inflammatory condition such as uveitis and in cataract which is chronic, compared to healthy controls. Methods: The TAC assay was done spectrophotometrically in the serum of Eales’ disease cases (n=20) as well as in other ocular pathologies involving oxidative stress namely, uveitis and cataract (n=20 each). The oxidative stress measured in terms of TBARS, was correlated with the TAC. Individual antioxidants namely vitamin C, E and glutathione were also estimated and correlated with TAC. Results: TAC was found to be significantly lower in Eales’ disease with active vasculitis (0.28 ± 0.09 mM, P<0.001), Eales’ disease with healed vasculitis (0.67 ± 0.09 mM), uveitis (0.46 ± 0.09 mM, P<0.001) and cataract (0.53 ± 0.1 mM, P=0.001) compared to the healthy controls, with a TAC level of 0.77 ± 0.09 mM. The TAC was found to correlate positively with vitamin E levels (P=0.05), GSH (P=0.02) but not with vitamin C, as seen in ED cases. In ED cases supplemented with vitamin E and C, there was a significant increase in the TAC level (P=0.02). Interpretation & conclusions: The TAC measurement provided a comprehensive assay for establishing a link between the antioxidant capacity and the risk of disease as well as monitoring antioxidant therapy. This method is a good substitute for assay of individual antioxidants as it clearly gives the status of the oxidative stress in the disease process.

Targeting CYP450 modulation to decrease the risk of induced cataract in the experimental model.

Background: Diabetes is one of the major causes of cataract. Some drugs prescribed for the treatment of diabetes are the modulators of CYP450, which may alter the risk of cataract. Objective: To study the effect of CYP450 modulation in galactosemic cataract. Materials and Methods: Male Sprague-Dawley suckling rats were allotted to four groups (n = 6), as follows: Group 1: Normal control, Group 2: Galactose control, Group 3: CYP450 inhibitor pretreated and Group 4: CYP450 inducer pretreated. Cataract was induced in animals of all groups except group 1 by feeding them galactose (50%), 21 days after parturition. From the eighteenth day of life, CYP450 inhibitor (nifedipine; 8.1 mg/kg) and CYP450 inducer (pioglitazone; 3.8 mg/kg) were given orally to groups 3 and 4, respectively. The maturation pattern of the cataract was observed by an operating microscope, every third day. Biochemical changes in the lenses of all groups, for example, CYP450 activity expressed as µM NADPH oxidized / unit time, alterations in the levels of total proteins, soluble proteins, and reduced glutathione (GSH) following the induction of cataract, were estimated. Results: The microscopic examination of the lenses indicated that CYP450 inhibitor pre-treatment delayed (fourteenth day) the occurrence of cataract, while CYP450 inducer pretreatment demonstrated an early (ninth day) cataract as compared to galactose control rats (twelfth day). A significant decrease and increase in CYP450 activity was observed with the CYP450 inhibitor and inducer pre-treatment, respectively. There was no alteration in the GSH level, but a significant increase in total and soluble protein was found in groups 3 and 4 as compared to group 2. Conclusion: CYP450 may have a role in the initiation of cataract without any effect on the maturation pattern, as revealed by the delayed occurrence of cataract with the CYP450 inhibitor and an early onset of cataract with the CYP450 inducer.

Preventive effect of onion juice on selenite-induced experimental cataract.

Purpose: To evaluate the effects of onion juice on sodium-selenite induced cataract formation. Materials and Methods: Thirty-two 10-day-old Wistar-albino rat pups were divided into four equal groups. Group 1 received only subcutaneous saline injection. In Group 2, sodium-selenite (30 nmol / g body weight) was injected subcutaneously. In Group 3, subcutaneous sodium-selenite was injected and one drop 50% diluted fresh juice of crude onion was instilled every 8 h into the right eye for 14 days; the left eye received no treatment. Group 4 rats were similar to those of Group 3, the only difference being that of undiluted fresh juice of crude onion. The development of cataract was assessed. Rat lenses were analyzed for total antioxidant (TA) level, and for activities of glutathione peroxidase (GPX) and superoxide dismutase (SOD). Results: Both eyes of all rats in Group 1 did not exhibit cataract formation . In Group 2, all rats developed Grade 3 cataract in the lenses of both eyes. The difference in exhibited cataract in the lens of the right eyes in all rats between Group 2 and any eyes of groups 3 or 4 were significant ( P = 0.001). The mean TA level and mean activities of SOD and GPX in Group 2 rat lenses were significantly lower than the values in lenses of all rats in Group 1 ( P = 0.001, 0.003, 0.001), and in the lenses of the right eyes of rats in Groups 3 and 4 ( P = 0.001, 0.020, 0.001). Conclusion: Instillation of onion juice into the rat eyes can effectively prevent selenite-induced cataract formation. This effect was associated with increased TA level, SOD and GPX activities in the lens.

The Efficacy of an Acrylic Intraocular Lens Surface Modified with Polyethylene Glycol in Posterior Capsular Opacification

To investigate if the surface modification of intraocular lens (IOL) is efficient in the prevention of posterior capsular opacification (PCO), the acrylic surface of intraocular lens (Acrysof(R)) was polymerized with polyethylene glycol (PEG-IOL). The human lens epithelial cells (1x10(4) cells/mL) were inoculated on PEG grafted or unmodified acrylic lenses for the control. The adherent cells on each IOL surface were trypsinized and counted. The every PEG-IOL was implanted in 20 New Zealand rabbits after removal of crystalline lens. The formations of PCO were checked serially through retroilluminated digital photography, and the severity scores were calculated using POCOman(R). The cell adherence patterns on each IOL were examined by scanning electron microscopy. As a result, the mean number of adherent cells of PEG-IOL (3.2+/-1.1x10(3)) tended to be smaller than that of the acrylic controls (3.6+/-1.9x10(3)) without a statistical significance (p=0.73). However, the mean severity of PCO formation in PEG-IOL was significantly lower than that in the control during the third to sixth weeks after surgery. Scanning electron microscopy revealed that the more patch-like cells were found firmly attached to the IOL surface in control than in the PEG-IOL. Conclusively, PEG polymerization to the acrylic IOL would possibly lessen the formation of PCO after cataract removal.

Studies on protein and taurine in normal, senile and diabetic cataractous human lenses.

An attempt has been made to explore the possible role of Taurine in cataractogenesis. Normal lenses were obtained from eye bank donors and cataractous lenses from patients who had undergone surgery for cataract extraction. Lenses were weighed and homogenised. Extraction, isolation and estimation of protein and taurine were carried out. It has been found that the lens wet weight increased progressively with the stage of maturation of cataract, i.e., from mature to hypermature which was significant and also with increase in age. Diabetic cataract group also showed an increase similar to that of senile cataract. Taurine and total protein decreases with different stages of maturation of cataract but not with age. It may be suggested that in the process of development of human senile cataract, there is (a) alteration in the structural integrity and permeability of lens membrane to protein and amino acids including taurine, (b) changes in the lens function including possible inhibition of proteins and amino acids (taurine) synthesis and transport across the cell membrane.

Possible Role of Amyloid beta- (1-40) -BSA Conjugates in Transdifferentiation of Lens Epithelial Cells

We investigated whether amyloid beta (Abeta) aggregates have transforming growth factor beta- like cytokine activity and cause transdifferention of lens epithelial cells, leading to certain types of cataract. In order to mimic Abetaaggregates, Abeta- (1-40) was crosslinked to bovine serum albumin (BSA) with disuccinimidyl suberate according to a previously described procedure. When human lens epithelial B-3 (HLE B-3) cells were treated with the Abeta- (1-40) -BSA conjugates, we observed the translocation of Smad-3, as well as the induced mRNA levels of fibronectin (FN), collagen type I (Col I), smooth muscle actin (SMA) and matrix metalloproteinase-2 (MMP-2). In addition, we investigated the morphology of rat whole lens cultured for 5 days in the presence of Abeta- (1-40) -BSA, and the immunohistochemical localizations of Abeta- (1-40) /amyloid precursor protein (APP) in human clinical tissues beneath the anterior capsules. In rat whole lens cultures, treatment with Abeta- (1-40) -BSA produced a transformed morphology that had multiple layers of lens epithelial cells. To compare the anterior capsules in anterior subcapsular cataracts with those in nuclear cataracts, immunohistochemical studies of Abeta/APP in human clinical tissues revealed that the predominant immunostaining of Abeta occurs in the anterior epithelial plaques, which likely produces the abnormal extracellular matrix. Thus, these findings suggest that Abeta aggregates in vivo are possibly involved in the regulatory process by which lens epithelial cells may transdifferentiate into fibroblast-like cells, as well as help understand the mechanisms which lead to certain types of cataractogenesis.

Selenite cataract and its attenuation by vitamin E in Wistar rats.

PURPOSE: To study the role of vitamin E in preventing cataract formation in experimental animals. METHODS: An experimental model (selenite cataract) was selected for this study. Selenite cataract was produced in rats by subcutaneous administration of sodium selenite. Biochemical and histological changes following induction of selenite cataract in weanling Wistar rats were studied vis-a-vis the role of vitamin E in attenuating or preventing cataractogenesis. RESULTS: Vitamin E was capable of preventing selenite cataractogenesis. Selenite cataract did not develop in 91.6% (11 of 12) and 76.7% (8 of 12) vitamin E treated rats, when administered on the 12th and 10th post partum day respectively. CONCLUSION: The study confirmed that selenite induced cataract in Wistar rats is attenuated by vitamin E.

Peróxido de hidrógeno en el humor acuoso: 1992-1997 / Hydrogen peroxide in the aqueous humor: 1992-1997

Ideas prevailing in 1991 on hydrogen peroxide in the aqueous humor are outlined. They are critically examined under the light of our finding that the method used to establish aqueous humor levels of peroxide generates itself peroxide during the short time span of the analysis. This is due to the fact that the probe used, dichlorophenol indophenol (DCPIP), spontaneously auto-oxidizes in the presence of oxygen. It was concluded then that the level of hydrogen peroxide in the aqueous humor cannot be higher than about 0.3 microM, the detection limit of the DCPIP method. It was also concluded that the statement commonly made in the literature that aqueous humor hydrogen peroxide derives from the oxidation of ascorbate, an abundant component of that fluid, is based solely on the use of the DCPIP method, and so could easily be due to a methodological artifact. The same applies to the statement that the levels of hydrogen peroxide are very high in human senile cataracts. The surprising resistance to accept the results and conclusions of our 1992 publication is documented. Finally, the content is discussed of an oral presentation made at the 1997 ARVO Annual Meeting in which an important portion of our results and conclusions was confirmed, perhaps signaling a shift towards a wider acceptance of our findings.

Flavin nucleotides in human lens: regional distribution in brunescent cataracts.

The biochemical mechanism(s) underlying brunescent cataracts remain unclear. Oxidative stress due to reactive oxygen species may have a role in the pigmentation process in eye lens. We have analysed human cataractous lenses for flavins by high-performance liquid chromatography (HPLC), since flavins are light sensitive and act as endogenous sensitizers generating reactive oxygen species in the eye. The most significant observation in this study is that higher levels of flavin nucleotides occur in brown lens compared to yellow lens. The concentration of flavin nucleotides (flavin monouncleotide, FMN + flavin adenine dinucleotide, FAD) was highest in the nuclear region of the lens followed by the cortical and capsule-epithelial regions. However, the ratio of FAD/FMN was lowest in the nuclear region of the lens followed by other regions. On the other hand, riboflavin was not detected in any of the lens (cataractous) regions. These results suggest that the observed increase in flavin nucleotides in the ocular tissue could contribute towards deepening of lens pigmentation.

Human lens epithelial layer in cortical cataract.

Normal and cataractous human eye lenses were studied by morphology and protein analysis. A marked decrease in protein sulfhydryl (PSH) and nonprotein sulfhydryl (NSPH) was observed in nuclear and cortical cataractous epithelia. Moreover, decrease in PSH contents and an increase in insoluble proteins were found to be correlated only in cortical cataractous epithelium which is also accompanied by various morphological abnormalities. In nuclear cataractous epithelium, however, there was very little insolubilisation of proteins. The epithelial morphology in nuclear cataracts was almost similar to normal lens epithelium. Hence, it is assumed that the protein insolubilisation and various morphological abnormalities are characteristics of cortical cataractous epithelium. This leads us to believe that opacification in cortical cataract might initiate in the epithelial layer.

Myoinositol and peroxidation--an in vitro study on human cataractous lens and human erythrocytes.

The effect of myoinositol on in vitro peroxidation induced by hydrogen peroxide in human erythrocytes and human cataractous lenses has been investigated. The lipid peroxidation was monitored as levels of thio barbituric reacting substances (TBARS). Addition of myoinositol decreased the peroxidation effect of hydrogen peroxide in a dose dependent manner. The results suggest a new antioxidant property for inositol.

Light scattering for young human and cataractous lenses.

The Rayleigh ratio for young human and cataractous lenses, having spherical particles with constant diameter embedded in a medium with a different refractive index, has been calculated as a function of concentration (volume fraction) using the theory of the small-q behaviour of the static structure factor, S(q). It involves treating the long-range forces between particles in the gel in the random phase approximation, dividing the pairwise interaction potential into a reference part and a perturbed part based on WCA model, and applying the perturbation approach of WCA to lenses obeying van der Waals' potential as a perturbative attraction over the Percus-Yevick (PY) hard sphere model. The calculated Rayleigh ratios are found in excellent agreement with experimental values, thereby showing that the present model works well for young human and cataractous lenses.

Increased glycosylation of human lens epithelial basement membrane in diabetes mellitus

We studied the nonenzymatic glycosylation of lens epithelial basement membranes (LEBM) of senile cataractous lenses of both diabetic and nondiabetic patients. The human LEBMs were isolated from surgically removed senile cataracts and purified by osmotic lysis and detergent treatments. Glycosylation assay of LEBMs was done using the colorimetric method of Fluckiger and Winterhalter. The glycosylation value ranged from 16.39 to 92.56 n mol/mg protein overall, with a mean of 63.54 +/- 24.56 n mol/mg protein for the diabetic specimens and a mean of 29.97 +/- 14.48 n mol/mg protein for the nondiabetic controls (P = 0.009). The study confirms our previous observation of in vivo glycosylation of the LEBM and further establishes that diabetic patients have a twofold increase in the amount of LEBM glycosylation when compared to their nondiabetic counterparts.

Increased glycosylation of human lens epithelial basement membrane in diabetes mellitus

We studied the nonenzymatic glycosylation of lens epithelial basement membranes (LEBM) of senile cataractous lenses of both diabetic and nondiabetic patients. The human LEBMs were isolated from surgically removed senile cataracts and purified by osmotic lysis and detergent treatments. Glycosylation assay of LEBMs was done using the colorimetric method of Fluckiger and Winterhalter. The glycosylation value ranged from 16.39 to 92.56 n mol/mg protein overall, with a mean of 63.54 +/- 24.56 n mol/mg protein for the diabetic specimens and a mean of 29.97 +/- 14.48 n mol/mg protein for the nondiabetic controls (P = 0.009). The study confirms our previous observation of in vivo glycosylation of the LEBM and further establishes that diabetic patients have a twofold increase in the amount of LEBM glycosylation when compared to their nondiabetic counterparts.

Acid phosphatase and lipid peroxidation in human cataractous lens epithelium.

The anterior lens epithelial cells undergo a variety of degenerative and proliferative changes during cataract formation. Acid phosphatase is primarily responsible for tissue regeneration and tissue repair. The lipid hydroperoxides that are obtained by lipid peroxidation of polysaturated or unsaturated fatty acids bring about deterioration of biological membranes at cellular and tissue levels. Acid phosphatase and lipid peroxidation activities were studied on the lens epithelial cells of nuclear cataract, posterior subcapsular cataract, mature cataract, and mixed cataract. Of these, mature cataractous lens epithelium showed maximum activity for acid phosphatase (516.83 moles of p-nitrophenol released/g lens epithelium) and maximum levels of lipid peroxidation (86.29 O.D./min/g lens epithelium). In contrast, mixed cataractous lens epithelium showed minimum activity of acid phosphatase (222.61 moles of p-nitrophenol released/g lens epithelium) and minimum levels of lipid peroxidation (54.23 O.D./min/g lens epithelium). From our study, we correlated the maximum activity of acid phosphatase in mature cataractous lens epithelium with the increased areas of superimposed cells associated with the formation of mature cataract. Likewise, the maximum levels of lipid peroxidation in mature cataractous lens epithelium was correlated with increased permeability of the plasma membrane. Conversely, the minimum levels of lipid peroxidation in mixed cataractous lens epithelium makes us presume that factors other than lipid peroxidation may also account for the formation of mixed type of cataract.

Cations, oxidants, light as causative agents in senile cataracts

Lens transparency is a function of regular cell shape, regular cell volume, minimal extracellular space, and minimal scatter elements. The cellular structure and molecular structure of the lens is reviewed. The importance of the cytoarchitecture especially the sutures, is discussed. The high cholesterol: phospholipid ratio of the lens fiber cell membranes is related to the functions of low permeability, low fluidity, and mechanical stability. Also reviewed are the contributions of the lens crystallins to lens clarity and to lens refractive index. The importance of intracellular and extracellular cation and water concentrations are reviewed. Finally the effects of systemic diseases, oxidation, and light on lens clarity are discussed relative to changes in lens fiber cell cation concentrations