Inhibition of Connective Tissue Growth Factor Expression in Adult Retinal Pigment Epithelial-19 Cells by Blocking Yes-Associated Protein/Transcriptional Coactivator with PDZ-Binding Motif Activity.

Purpose: To investigate the effect of yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) on connective tissue growth factor (CTGF) expression in adult retinal pigment epithelial (ARPE)-19 cells. We also studied the inhibitory effect of K-975, a new pan-transcriptional enhanced associate domain (TEAD) inhibitor, and luteolin, a plant-derived flavonoid on CTGF expression. Methods: ARPE-19 cells were transfected with either YAP or TAZ overexpression plasmid or treated with transforming growth factor (TGF)-ß2. The cells were cultured either with or without K-975 or luteolin. The expression of YAP, TAZ, and CTGF was examined using real-time PCR. Results: ARPE-19 cells overexpressing YAP or TAZ exhibited significantly increased CTGF expression. This increase was attenuated by K-975 or luteolin alone. TGF-ß2 treatment significantly raised the expression of not just YAP and TAZ, but also CTGF in ARPE-19 cells. TGF-ß2 treatment-enhanced CTGF expression was considerably lowered by the addition of K-975 or luteolin. Conclusions: Overexpression of YAP or TAZ and treatment with TGF-ß2 led to an increase in the expression of CTGF in ARPE-19 cells. These increases were attenuated by treatment with K-975 and luteolin. These findings suggest that YAP and TAZ may be related to the expression of CTGF in ARPE-19 cells and that K-975 and luteolin can be explored as potential therapeutic agents for preventing CTGF production in vitreoretinal fibrosis.

Antiresorptive agent-related osteonecrosis of the jaw risk in cancer patients before bone-modifying agent therapy: A retrospective study of 511 patients.

INTRODUCTION: Antiresorptive agent-related osteonecrosis of the jaw (ARONJ) is a serious adverse event associated with therapies for advanced cancer involving bone-modifying agents (BMAs). Although many studies have investigated the risk factors for ARONJ, none have provided sufficient clarifications. We aimed to determine whether there is a correlation between tooth extraction before BMA therapy and the development of ARONJ. PATIENTS AND METHODS: We included 511 patients who were admitted to our department before BMA therapy. The incidence of ARONJ and its risk factors was retrospectively evaluated, focusing on tooth extraction before BMA therapy and radiological findings of the extracted tooth. RESULTS: Of the 511 patients, 135 underwent tooth extraction before undergoing BMA therapy. ARONJ was observed in 17.7 % and 7.2 % of patients in the extraction and non-extraction groups, respectively (p = 0.0002). Regarding the findings of panoramic radiographs before tooth extraction, the incidence of periapical disease was significantly higher in the ARONJ group than that in the non-ARONJ group (50 % and 27 %, respectively, p = 0.034), and the proportion of bone sclerotic changes in the surrounding teeth was significantly higher in the ARONJ group (58.3 %) than in the non-ARONJ group (24.4 %) (p = 0.0004). CONCLUSION: The presence of a hopeless tooth that requires extraction prior to BMA therapy is a risk factor for MRONJ. However, extraction of such teeth should be considered, as this is known to decrease the risk of MRONJ development after BMA therapy initiation. Moreover, the radiological findings of periapical disease and bone sclerotic changes in the extracted teeth identified in this study can be used as novel predictive factors for ARONJ.

Mizagliflozin, a selective SGLT1 inhibitor, improves vascular cognitive impairment in a mouse model of small vessel disease.

Previously, we showed that sodium/glucose cotransporter 1 (SGLT1) participates in vascular cognitive impairment in small vessel disease. We hypothesized that SGLT1 inhibitors can improve the small vessel disease induced-vascular cognitive impairment. We examined the effects of mizagliflozin, a selective SGLT1 inhibitor, and phlorizin, a non-selective SGLT inhibitor, on vascular cognitive impairment in a mouse model of small vessel disease. Small vessel disease was created using a mouse model of asymmetric common carotid artery surgery (ACAS). Two and/or 4 weeks after ACAS, all experiments were performed. Cerebral blood flow (CBF) was decreased in ACAS compared with sham-operated mice. Phlorizin but not mizagliflozin reversed the decreased CBF of ACAS mice. Both mizagliflozin and phlorizin reversed the ACAS-induced decrease in the latency to fall in a wire hang test of ACAS mice. Moreover, they reversed the ACAS-induced longer escape latencies in the Morris water maze test of ACAS mice. ACAS increased SGLT1 and proinflammatory cytokine gene expressions in mouse brains and phlorizin but not mizagliflozin normalized all gene expressions in ACAS mice. Hematoxylin/eosin staining demonstrated that they inhibited pyknotic cell death in the ACAS mouse hippocampus. In PC12HS cells, IL-1ß increased SGLT1 expression and decreased survival rates of cells. Both mizagliflozin and phlorizin increased the survival rates of IL-1ß-treated PC12HS cells. These results suggest that mizagliflozin and phlorizin can improve vascular cognitive impairment through the inhibition of neural SGLT1 and phlorizin also does so through the improvement of CBF in a mouse model of small vessel disease.

Effect of Geranylgeranylacetone on Ultraviolet Radiation Type B-Induced Cataract in Heat-Shock Transcription Factor 1 Heterozygous Mouse.

PURPOSE: We investigated whether heat-shock transcription factor 1 (HSF1) was involved in ultraviolet radiation type B (UVR-B)-induced lens opacity (cataract) using HSF1 heterozygous mice. We also examined the effects of geranylgeranylacetone (GGA), an inducer of heat-shock proteins via activation of HSF, on the UVR-B-induced cataract. MATERIAL AND METHODS: Male HSF1+/- and WT mice were unilaterally exposed to UVR-B (total: 1200mJ) at 16 weeks of age. At 48 h after the last UVR-B irradiation, the lens was isolated and the induction of the cataract was quantified as the cataract area ratio (opacity area/anterior capsule). GGA was orally administered at a dosage of 500 mg/kg once a day for two days before the first UVR-B exposure until the end of the experiment (21days in total). RESULTS: The HSF1 expression was more greatly decreased in the lens from HSF1+/- mice than in that from WT mice (p < 0.01). UVR-B exposure could mainly induce cataracts in the anterior capsule in both HSF1+/- and WT mice, while the opacity of the lens was markedly enhanced in HSF1+/- mice compared to that in WT mice(p (0.01). GGA treatment could prevent the induction of lens opacity by UVR-B exposure in both WT and HSF1+/- mice as compared with the non-administration group (p < 0.01). No obvious alteration by the UVR-B radiation was seen in lens protein levels of αA-crystallin, αB-crystallin, or γ-crystallin with or without GGA administration among all groups of mice. In contrast to the crystallins, the lens protein level of HSP25 was decreased by UVR-B exposure in both HSF1+/- and WT mice, and was significantly recovered in WT mice by the GGA treatment (p < 0.01). The induction of HSP25 was suppressed in HSF1+/- mice compared with that in WT mice. CONCLUSIONS: These data suggest that HSF1 plays an important role in the occurrence of UVR-B-induced cataracts, possibly via regulation of HSPs such as HSP25.

Upregulation of inorganic pyrophosphatase 1 as a JNK phosphatase in hypothyroid embryonic chick cerebellum.

AIM: Thyroid hormones play important roles in vertebrate neuronal development and differentiation. In our previous study, we showed that fetal thyroid dysfunction led to impaired social behaviors of hatchlings on post-hatch day 3, as well as to impaired learning and memory determined by the imprinting preference. However, little is known about the mechanisms underlying the direct adverse effects of fetal thyroid dysfunction on neuronal development. MATERIALS AND METHODS: We used a chick embryo as a fetal model to investigate the effects of prenatal exposure to antithyroid drugs on neuronal development in the chick cerebellum. Methimazole (MMI) at a dose of 20µmol/egg was administered to eggs on day 14, while the control was given only a vehicle. In order to address the underlying mechanisms of the impaired behavior, proteomic approaches were employed in the chick cerebellum two days after MMI treatment. KEY FINDINGS: In this experiment, we found that inorganic pyrophosphatase 1 (PPA1) was upregulated in the chick cerebellum treated with MMI, and we confirmed this upregulation of PPA1 by Western blot analysis as well as by RT-PCR analysis. Concomitant with the upregulation of PPA1, a marked reduction in JNK activity, as well as of phospho-JNK level, was detected in the MMI-treated chick cerebellum. SIGNIFICANCE: Since PPA1 can dephosphorylate JNK, these results suggest that the upregulation of PPA1 during neuronal development in the hypothyroid chick cerebellum may lead to impaired social behaviors as well as to impaired learning and memory via JNK dephosphorylation and inactivation in the chick cerebellum.

Effect of astaxanthin on cataract formation induced by glucocorticoids in the chick embryo.

PURPOSE: To examine whether astaxanthin (AST) prevent the cataract formation induced by glucocorticoid in chick embryo. MATERIALS AND METHODS: Hydrocortisone hemisuccinate sodium (HC) (0.5 µmol/egg) was administered directly into the air chamber in the egg shell of chick embryo day 15. The eggs were then kept in an incubator at same conditions and administered 100 µL of 50 (HC + AST50 group), 80 (HC + AST80 group), 100 (HC + AST100 group) mg/mL of AST solutions dissolved in dimethyl sulfoxide (DMSO) 3 h after administration of HC. In addition, non-HC treated group (treated with physiological saline without HC and 100 µL of DMSO), HC-alone group (treated with 0.5 µmol of HC and 100 µL of DMSO), and AST100 group (treated with physiological saline without HC and 100 µL of DMSO) were also incorporated. After 48 h of treatment, lenses were removed from embryo and classified into five stages according to developed opacity. The amounts of reduced glutathione in the lenses and the blood glucose levels were measured. RESULTS: The average scores of lens opacitiy were 2.63 ± 1.02 nmol/lens (HC-alone), 2.78 ± 0.97 nmol/lens (HC + AST50), 2.22 ± 1.20 nmol/lens (HC + AST80) and 1.84 ± 0.83 nmol/lens (HC + AST100; p < 0.05), respectively. Administration of AST decreased the lens opacity dose-dependently. The amounts of reduced glutathione in lenses were 11.6 ± 2.8 nmol/lens (HC-alone), 11.3 ± 2.7 nmol/lens (HC + AST50), 13.4 ± 2.4 nmol/lens (HC + AST80) and 13.7 ± 3.1 nmol/lens (HC + AST100; p < 0.05), respectively. Higher levels of AST prevented loss of reduced glutathione from the lens. CONCLUSION: These findings support that AST protects glucocorticoid-induced cataract in chick embryo.

Altered magnetic resonance images of brain and social behaviors of hatchling, and expression of thyroid hormone receptor ßmRNA in cerebellum of embryos after Methimazole administration.

RATIONALE AND OBJECTIVES: The effects of low thyroid hormone level during embryogenesis on MRI of the brain and social behaviors of hatchlings were examined using "fertilized hen's egg-embryo-chick" system. METHODS AND RESULTS: Control and hatchlings treated with methimazole (20 µmol/egg), which hatched 3 days later than controls were examined. The results are as follows: 1. The MRI examination of the midsagittal section of the brain on hatch day showed that the sizes, by T1- and ADC values by diffusion-weighted images, of the optic lobe and cerebellum of the MMI-hatchlings were significantly bigger than those of the controls. 2. The social behaviors on post-hatch day 3 were based on the following tests: (a) Aggregation test: The speed of four chicks, individually isolated by cardboard barriers in a box, to make a group upon the removal of barriers. (b) Belongingness tests: The speed of a chick isolated at a corner to join the group of three chicks placed at the opposite corner. (c) Vocalization test: The number of decibel produced by a chick isolated at a corner using a sound meter. These tests demonstrated that MMI-hatchlings took longer times and had weaker vocalization than the controls, significantly. 3. Upregulation of THRß mRNA after MMI treatment suggested that THR was necessary for cerebellum development. CONCLUSIONS: The MMI exposure during the last week of embryogenesis possibly delayed the myelination of certain brain regions and impaired the social behaviors of hatchlings. The chick embryos can be easily induced with hypothyroidism without maternal influences, and the hatchling's behaviors were analyzed using a video camera. The present method will be useful for assessing the effects of unfavorable influences during embryogenesis on social behaviors in later life.

Regulation of neurite growth by inorganic pyrophosphatase 1 via JNK dephosphorylation.

Neural cell differentiation during development is controlled by multiple signaling pathways, in which protein phosphorylation and dephosphorylation play an important role. In this study, we examined the role of pyrophosphatase1 (PPA1) in neuronal differentiation using the loss and gain of function analysis. Neuronal differentiation induced by external factors was studied using a mouse neuroblastoma cell line (N1E115). The neuronal like differentiation in N1E115 cells was determined by morphological analysis based on neurite growth length. In order to analyze the loss of the PPA1 function in N1E115, si-RNA specifically targeting PPA1 was generated. To study the effect of PPA1 overexpression, an adenoviral gene vector containing the PPA1 gene was utilized to infect N1E115 cells. To address the need for pyrophosphatase activity in PPA1, D117A PPA1, which has inactive pyrophosphatase, was overexpressed in N1E115 cells. We used valproic acid (VPA) as a neuronal differentiator to examine the effect of PPA1 in actively differentiated N1E115 cells. Si-PPA1 treatment reduced the PPA1 protein level and led to enhanced neurite growth in N1E115 cells. In contrast, PPA1 overexpression suppressed neurite growth in N1E115 cells treated with VPA, whereas this effect was abolished in D117A PPA1. PPA1 knockdown enhanced the JNK phosphorylation level, and PPA1 overexpression suppressed it in N1E115 cells. It seems that recombinant PPA1 can dephosphorylate JNK while no alteration of JNK phosphorylation level was seen after treatment with recombinant PPA1 D117A. Enhanced neurite growth by PPA1 knockdown was also observed in rat cortical neurons. Thus, PPA1 may play a role in neuronal differentiation via JNK dephosphorylation.

Phenotype of cardiomyopathy in cardiac-specific heat shock protein B8 K141N transgenic mouse.

A K141N missense mutation in heat shock protein (HSP) B8, which belongs to the small HSP family, causes distal hereditary motor neuropathy, which is characterized by the formation of inclusion bodies in cells. Although the HSPB8 gene causes hereditary motor neuropathy, obvious expression of HSPB8 is also observed in other tissues, such as the heart. The effects of a single mutation in HSPB8 upon the heart were analyzed using rat neonatal cardiomyocytes. Expression of HSPB8 K141N by adenoviral infection resulted in increased HSPB8-positive aggregates around nuclei, whereas no aggregates were observed in myocytes expressing wild-type HSPB8. HSPB8-positive aggresomes contained amyloid oligomer intermediates that were detected by a specific anti-oligomer antibody (A11). Expression of HSPB8 K141N induced slight cellular toxicity. Recombinant HSPB8 K141N protein showed reactivity against the anti-oligomer antibody, and reactivity of the mutant HSPB8 protein was much higher than that of wild-type HSPB8 protein. To extend our in vitro study, cardiac-specific HSPB8 K141N transgenic (TG) mice were generated. Echocardiography revealed that the HSPB8 K141N TG mice exhibited mild hypertrophy and apical fibrosis as well as slightly reduced cardiac function, although no phenotype was detected in wild-type HSPB8 TG mice. A single point mutation of HSPB8, such as K141N, can cause cardiac disease.

Impaired social behavior in chicks exposed to sodium valproate during the last week of embryogenesis.

RATIONALE AND OBJECTIVES: To evaluate direct exposure to sodium valproate (VPA) during embryogenesis, we administered VPA to chick embryos and examined their social behaviors after hatching. METHODS AND RESULTS: Embryos treated with VPA (35 µmol/egg) on day 14 were similar to controls for hatching date (day 21) and hatchlings' abilities, such as motor, imprinting, and surface righting. However, these VPA chicks on posthatching day 3 scored significantly low in the chick's social separation stress (SSS) test as follows. Aggregation test evaluated the speed of four chicks, individually isolated by a cardboard in a box, to aggregate upon removal of the cardboards. Belongingness test evaluated the speed of a chick isolated at a corner to join the group of three chicks placed at the opposite corner. Vocalization test for each chick was performed in an isolated corner by using a sound level meter. The results demonstrated that compared with controls, VPA chicks were significantly slow in aggregation (12.7 ± 2.5 s vs. 2.9 ± 0.9 s, p = 0.006) and belongingness (3.6 ± 0.28 s/40 cm vs. 2.6 ± 0.14 s/40 cm, P = 0.003) and weak in vocalization (13.4 ± 2.8 dB/30 s vs. 26.7 ± 1.3 dB/30 s, P = 0.001), respectively. Weight of cerebellum of VAP chick was 15 % lighter than controls (P = 0.004). CONCLUSIONS: Chick embryos exposed to VPA during the last week of embryogenesis had impaired social behaviors in spite of normal mortar and imprinting ability. The present method will be a useful animal model for assessing the effects of environment during embryogenesis on social behaviors in later life.

Lovastatin alters TGF-ß-induced epithelial-mesenchymal transition in porcine lens epithelial cells.

PURPOSE: To determine whether lovastatin affects the epithelial-mesenchymal transition (EMT) in porcine lens epithelial cells (LECs) induced by transforming growth factor-ß (TGF-ß). MATERIALS AND METHODS: Porcine LECs were cultured in Dulbecco's Modified Eagle Medium (DMEM) for 24 h. The cultured cells were then exposed or not exposed to lovastatin (10 µM) for 18 h and then stimulated with or not stimulated with TGF-ß2 (5 ng/ml) for 24 h. The expression of α-smooth muscle actin (α-SMA), a marker of myofibroblasts, was determined by real-time PCR, and the expression of α-SMA protein was determined by Western blot. The effect of lovastatin on the expression of the mRNA of collagen type 1 (COL1) was determined by real-time PCR. To assess cell contractility, LECs were cultured in collagen gel with or without pretreatment of lovastatin and exposure of TGF-ß2. The longest and shortest diameters of the gels were measured and the area was determined. RESULTS: Exposure of LECs to TGF-ß2 increased the expression of the mRNA and protein of α-SMA and the mRNA of COL1A1. TGF-ß2 increased the degree of contraction of collagen gel. These findings indicated that TGF-ß2 promoted EMT, and the pretreatment of the LECs with lovastatin blocked these changes induced by TGF-ß2. CONCLUSION: Lovastatin inhibits the TGF-ß-induced EMT of cultured porcine LECs. This suggests that lovastatin should be considered as a new agent to prevent postoperative complications associated with EMT of LECs.

Effect of vitamin C depletion on UVR-B induced cataract in SMP30/GNL knockout mice.

We investigated whether decreased vitamin C (VC) in a mouse model increases lens opacity (cataract) induced by in vivo exposure to ultraviolet radiation type B (UVR-B). Senescence marker protein-30 (SMP30) knockout (KO) mice, which cannot synthesize VC due to genetic disruption of the gluconolactonase (GNL) gene, were divided into 2 groups: VC sufficient (VC (+)) and VC deficient (VC (-)). Starting at 1 month of age, these groups had free access to water containing 0.0375 and 1.5 g/L of VC, respectively. SMP30 KO VC (-), SMP30 KO VC (+), and wild-type (WT) mice, all 14 weeks of age, were unilaterally exposed in vivo to UVR-B (200 mW/cm(2)) for 100 s twice a week for 3 weeks (total: 1200 mJ/cm(2)). At 48 h after the last UVR-B exposure, cataract morphology was documented, and the ratio of cataract induction was quantified as the cataract area ratio (opacity area/anterior capsule). UVR-B exposure induced cataract mainly at anterior sub-capsular in SMP30 KO VC (-), SMP30 KO VC (+), and WT mice. In SMP30 KO VC (-) lenses the opacities were more extensive than in SMP30 KO VC (+) or WT lenses (cataract area ratios: 59.3% ± 10% vs. 32.2% ± 11.7% or 29.0% ± 9.0%; P < 0.01). In conclusion, VC depletion may increase the susceptibility to develop UVR-B induced cataracts in mice unable to endogenously produce VC.

Effect of antithyroid drug on chick embryos during the last week of development: delayed hatching and decreased cerebellar acetylcholinesterase activity.

AIM: Hypothyroid state during embryogenesis disturbs normal growth and brain development, influencing later life. To evaluate the harmful consequences of the state during embryogenesis using an animal model, we inhibited thyroid hormone biosynthesis in chick embryos by using methimazole (MMI). MATERIAL AND METHODS: Typically, embryos were treated with MMI (20 µmol/egg) on day 14, and examined on specific days. RESULTS: Of the control embryos, 94% hatched on day 21, whereas 0% and 60% of MMI-treated embryos hatched on days 21 and 24, respectively. MMI retarded the rates of bodyweight gain as well as liver and heart development, and delayed hatching. However, the external differences in appearance and differences in the weights of the newly hatched control chicks on day 21 and the MMI-treated chicks on day 24 were less obvious. Embryos treated with MMI exhibited increased mass in their brain parts on day 24. Most notably, the treatment resulted in a 1.35-fold increase in cerebellum weight compared to that of the untreated animals. Acetylcholinesterase activity in the cerebellum on the day of hatching decreased significantly to 0.85-fold that of the untreated controls. Thyroid hormone receptor ß mRNA was detected from day 12 and dramatically expressed from day 19 to the day of hatching. CONCLUSION: The 'fertilized hen's egg-chick embryo-chick system' is an appropriate animal model for investigating the hypothyroid state during embryogenesis. Decreased cerebellar acetylcholinesterase activity after MMI treatment was assumed to relate to a mechanism of motor and cognitive deficits in congenital hypothyroidism.

Effects of glucocorticoid on brain acetylcholinesterase of developing chick embryos.

AIM: Fetal exposure to excessive or deficient glucocorticoids may alter the programming in differentiation and maturation of various tissues including the brain and nervous system, leading to dysfunctions later in life. For further exploration of this possibility, we established an animal model using developing chick embryos. METHODS: (i) Reverse-transcription polymerase chain reaction was used to determine the expression of glucocorticoid receptor mRNA in the brain of chick embryos. (ii) Embryos on day 15 were administered betamethasone or mifepristone and their cerebrum, cerebellum and optic lobe were investigated to determine the activity of acetylcholinesterase. RESULTS: (i) Glucocorticoid receptor mRNA was shown to be present in the cerebrum, cerebellum and optic lobe. (ii) After the administration of betamethasone, acetylcholinesterase activities in the cerebrum, cerebellum and optic lobe on day 19 were 1.5- to 2-fold higher than those of untreated control. Weights of body and brain parts were 0.65-0.75-fold relative to control values. However, these differences were less noticeable on day 22. (iii) Administration of mifepristone before treatment with betamethasone prevented high-dose betamethasone-induced changes in acetylcholinesterase activity and bodyweights on day 19. Administration of mifepristone alone did not induce differences from the control. CONCLUSIONS: The cerebrum, cerebellum and optic lobe of chick embryos could be influenced by glucocorticoids because of the presence of glucocorticoid receptor mRNA. Although the effects observed after treatment with excess glucocorticoids (even no effects after mifepristone treatment) were transitory, they may alter the developmental program in ways that could result in lasting change and influence behavioral activities after hatching.