Therapeutic strategies for glaucoma and optic neuropathies.

Glaucoma is a neurodegenerative eye disease that causes permanent vision impairment. The main pathological characteristics of glaucoma are retinal ganglion cell (RGC) loss and optic nerve degeneration. Glaucoma can be caused by elevated intraocular pressure (IOP), although some cases are congenital or occur in patients with normal IOP. Current glaucoma treatments rely on medicine and surgery to lower IOP, which only delays disease progression. First-line glaucoma medicines are supported by pharmacotherapy advancements such as Rho kinase inhibitors and innovative drug delivery systems. Glaucoma surgery has shifted to safer minimally invasive (or microinvasive) glaucoma surgery, but further trials are needed to validate long-term efficacy. Further, growing evidence shows that adeno-associated virus gene transduction and stem cell-based RGC replacement therapy hold potential to treat optic nerve fiber degeneration and glaucoma. However, better understanding of the regulatory mechanisms of RGC development is needed to provide insight into RGC differentiation from stem cells and help choose target genes for viral therapy. In this review, we overview current progress in RGC development research, optic nerve fiber regeneration, and human stem cell-derived RGC differentiation and transplantation. We also provide an outlook on perspectives and challenges in the field.

Promotion of Protein Solubility and Reduction in Stiffness in Human Lenses by Aggrelyte-1: Implications for Reversing Presbyopia.

With aging, human lenses lose the ability to focus on nearby objects due to decreases in accommodative ability, a condition known as presbyopia. An increase in stiffness or decrease in lens elasticity due to protein aggregation and insolubilization are the primary reasons for presbyopia. In this study, we tested aggrelyte-1 (S,N-diacetyl glutathione diethyl ester) for its ability to promote protein solubility and decrease the stiffness of lenses through its dual property of lysine acetylation and disulfide reduction. Treatment of water-insoluble proteins from aged human lenses (58-75 years) with aggrelyte-1 significantly increased the solubility of those proteins. A control compound that did not contain the S-acetyl group (aggrelyte-1C) was substantially less efficient in solubilizing water-insoluble proteins. Aggrelyte-1-treated solubilized protein had significant amounts of acetyllysine, as measured by Western blotting and LC-MS/MS. Aggrelytes increased the protein-free thiol content in the solubilized protein. Aged mouse (7 months) and human (44-66 years) lenses treated with aggrelyte-1 showed reduced stiffness accompanied by higher free thiol and acetyllysine levels compared with those treated with aggrelyte-1C or untreated controls. Our results suggested that aggrelyte-1 reduced lens stiffness through acetylation followed by disulfide reduction. This proof-of-concept study paves the way for developing aggrelyte-1 and related compounds to reverse presbyopia.

Aggrelyte-2 promotes protein solubility and decreases lens stiffness through lysine acetylation and disulfide reduction: Implications for treating presbyopia.

Aging proteins in the lens become increasingly aggregated and insoluble, contributing to presbyopia. In this study, we investigated the ability of aggrelyte-2 (N,S-diacetyl-L-cysteine methyl ester) to reverse the water insolubility of aged human lens proteins and to decrease stiffness in cultured human and mouse lenses. Water-insoluble proteins (WI) of aged human lenses (65-75 years) were incubated with aggrelyte-2 (500 µM) for 24 or 48 h. A control compound that lacked the S-acetyl group (aggrelyte-2C) was also tested. We observed 19%-30% solubility of WI upon treatment with aggrelyte-2. Aggrelyte-2C also increased protein solubility, but its effect was approximately 1.4-fold lower than that of aggrelyte-2. The protein thiol contents were 1.9- to 4.9-fold higher in the aggrelyte-2- and aggrelyte-2C-treated samples than in the untreated samples. The LC-MS/MS results showed Nε -acetyllysine (AcK) levels of 1.5 to 2.1 nmol/mg protein and 0.6 to 0.9 nmol/mg protein in the aggrelyte-2- and aggrelyte-2C-treated samples. Mouse (C57BL/6J) lenses (incubated for 24 h) and human lenses (incubated for 72 h) with 1.0 mM aggrelyte-2 showed significant decreases in stiffness with simultaneous increases in soluble proteins (human lenses) and protein-AcK levels, and such changes were not observed in aggrelyte-2C-treated lenses. Mass spectrometry of the solubilized protein revealed AcK in all crystallins, but more was observed in α-crystallins. These results suggest that aggrelyte-2 increases protein solubility and decreases lens stiffness through acetylation and disulfide reduction. Aggrelyte-2 might be useful in treating presbyopia in humans.

AAV2-Mediated Expression of HspB1 in RGCs Prevents Somal Damage and Axonal Transport Deficits in a Mouse Model of Ocular Hypertension.

Purpose: Ocular hypertension is a significant risk factor for vision loss in glaucoma caused by the death of retinal ganglion cells (RGCs). We investigated whether small heat shock proteins (sHsps) expressed in RGCs protect those cells against ocular hypertension in mice. Methods: AAV2 vectors encoding genes for one of the following four human sHsps: HSPB1, HSPB4, HSPB5, or HSPB6 were constructed for RGC-specific expression. Ischemia/reperfusion was induced by elevating the intraocular pressure (IOP) to 120 mm Hg for one hour, followed by a rapid return to normal IOP. Microbeads (MB) were injected into the anterior chamber of mice to induce ocular hypertension. RGC death and glial activation were assessed by immunostaining for Brn3a, RBPMS, Iba1, and glial fibrillary acid protein in retinal flat mounts. RGC axonal defects were evaluated by anterograde transport of intravitreally injected cholera toxin-B. RGC function was assessed by pattern electroretinography. Results: Among the sHsps, HspB1 offered the best protection against RGC death from ischemia/reperfusion injury in the mouse retina. Intravitreal administration of AAV2-HSPB1 either two weeks before or one week after instituting ocular hypertension resulted in significant prevention of RGC loss. The MB-injected mice showed RGC axonal transportation defects, but AAV2-HSPB1 administration significantly inhibited this defect. AAV2-HSPB1 prevented glial activation caused by ocular hypertension. More importantly, a single injection of AAV2-HSPB1 protected RGCs long-term in MB-injected eyes. Conclusions: The administration of AAV2-HSPB1 inhibited RGC death and axonal transport defects and reduced glial activation in a mouse model of ocular hypertension. Translational Relevance: Our results suggested that the intravitreal delivery of AAV2-HSPB1 could be developed as a gene therapy to prevent vision loss on a long-term basis in glaucoma patients.

Peptains block retinal ganglion cell death in animal models of ocular hypertension: implications for neuroprotection in glaucoma.

Ocular hypertension is a significant risk factor for vision loss in glaucoma due to the death of retinal ganglion cells (RGCs). This study investigated the effects of the antiapoptotic peptides peptain-1 and peptain-3a on RGC death in vitro in rat primary RGCs and in mouse models of ocular hypertension. Apoptosis was induced in primary rat RGCs by trophic factor deprivation for 48 h in the presence or absence of peptains. The effects of intravitreally injected peptains on RGC death were investigated in mice subjected to retinal ischemic/reperfusion (I/R) injury and elevated intraocular pressure (IOP). I/R injury was induced in mice by elevating the IOP to 120 mm Hg for 1 h, followed by rapid reperfusion. Ocular hypertension was induced in mice by injecting microbeads (MB) or silicone oil (SO) into the anterior chamber of the eye. Retinal flatmounts were immunostained with RGC and activated glial markers. Effects on anterograde axonal transport were determined by intravitreal injection of cholera toxin-B. Peptain-1 and peptain-3a inhibited neurotrophic factor deprivation-mediated RGC apoptosis by 29% and 35%, respectively. I/R injury caused 52% RGC loss, but peptain-1 and peptain-3a restricted RGC loss to 13% and 16%, respectively. MB and SO injections resulted in 31% and 36% loss in RGCs following 6 weeks and 4 weeks of IOP elevation, respectively. Peptain-1 and peptain-3a inhibited RGC death; the loss was only 4% and 12% in MB-injected eyes and 16% and 15% in SO-injected eyes, respectively. Anterograde transport was defective in eyes with ocular hypertension, but this defect was substantially ameliorated in peptain-injected eyes. Peptains suppressed ocular hypertension-mediated retinal glial activation. In summary, our results showed that peptains block RGC somal and axonal damage and neuroinflammation in animal models of glaucoma. We propose that peptains have the potential to be developed as therapeutics against neurodegeneration in glaucoma.

Lactococcus lactis KF140 Reduces Dietary Absorption of Nε - (Carboxymethyl)lysine in Rats and Humans via ß-Galactosidase Activity.

Background and Aims: Excessive intake of advanced glycation end products (AGEs), which are formed in foods cooked at high temperatures for long periods of time, has negative health effects, such as inflammatory responses and oxidative stress. Nε-(Carboxymethyl)lysine (CML) is one of the major dietary AGEs. Given their generally recognized as safe status and probiotic functionalities, lactic acid bacteria may be ideal supplements for blocking intestinal absorption of food toxicants. However, the protective effects of lactic acid bacteria against dietary AGEs have not been fully elucidated. Materials and Methods: We investigated the effect of treatment with Lactococcus lactis KF140 (LL-KF140), which was isolated from kimchi, on the levels and toxicokinetics of CML. The CML reduction efficacies of the Lactococcus lactis KF140 (LL-KF140), which was isolated from kimchi, were conducted by in vitro test for reducing CML concentration of the casein-lactose reaction product (CLRP) and in vivo test for reducing serum CML level of LL-KF140 administered rats at 2.0 × 108 CFU/kg for14 days. In addition, 12 volunteers consuming LL-KF140 at 2.0 × 109 CFU/1.5 g for 26 days were determined blood CML concentration and compared with that before intake a Parmesan cheese. Results: Administration of LL-KF140 reduced serum CML levels and hepatic CML absorption in rats that were fed a CML-enriched product. In a human trial, the intake of LL-KF140 prevented increases in the serum levels of CML and alanine aminotransferase after consumption of a CML-rich cheese. LL-KF140 was determined to presence in feces through metagenome analysis. Furthermore, ß-galactosidase, one of the L. lactis-produced enzymes, inhibited the absorption of CML and reduced the levels of this AGE, which suggests an indirect inhibitory effect of LL-KF140. This study is the first to demonstrate that an L. lactis strain and its related enzyme contribute to the reduction of dietary absorption of CML.

Small Heat Shock Proteins in Retinal Diseases.

This review summarizes the latest findings on small heat shock proteins (sHsps) in three major retinal diseases: glaucoma, diabetic retinopathy, and age-related macular degeneration. A general description of the structure and major cellular functions of sHsps is provided in the introductory remarks. Their role in specific retinal diseases, highlighting their regulation, role in pathogenesis, and possible use as therapeutics, is discussed.

Methylglyoxal-Derived Advanced Glycation End Products (AGE4) Promote Cell Proliferation and Survival in Renal Cell Carcinoma Cells through the RAGE/Akt/ERK Signaling Pathways.

Advanced glycation end products (AGEs) are the products formed through a non-enzymatic reaction of reducing sugars with proteins or lipids. There is a potential for toxicity in the case of AGEs produced through glycation with dicarbonyl compounds including methylglyoxal, glyoxal, and 3-deoxyglucosone. The AGEs bind the receptor for advanced glycation end products (RAGE) and stimulate the mitogen-activated protein (MAP) kinase signaling pathway that can increase the production of matrix metalloproteinases (MMPs). In addition, AGE-induced protein kinase B (Akt) signaling can promote cancer cell proliferation and contribute to many diseases such as kidney cancer. In light of the lack of extensive study of the relationship between methylglyoxal-induced AGEs (AGE4) and renal cancer, we studied the proliferous and anti-apoptotic effects of AGE4 on renal cell carcinoma (RCC) in this study. AGE4 treatment was involved in the proliferation and migration of RCC cells in vitro by upregulating proliferating cell nuclear antigen (PCNA) and MMPs while suppressing apoptotic markers such as Bax and caspase 3. Moreover, Akt and extracellular-signal-regulated kinase (ERK) were phosphorylated in RCC cells with AGE4 treatment. As a result, this study demonstrated that AGE4-RAGE axis can promote the growth ability of RCC by inducing PCNA, MMPs, and inhibiting apoptosis in RCC via the Akt and ERK signaling pathways.

Transforming growth factor-ß2-mediated mesenchymal transition in lens epithelial cells is repressed in the absence of RAGE.

Transforming growth factor-ß2 (TGFß2)-mediated epithelial to mesenchymal transition (EMT) in lens epithelial cells (LECs) has been implicated in fibrosis associated with secondary cataracts. In this study, we investigated whether the receptor for advanced glycation end products (RAGE) plays a role in TGFß2-mediated EMT in LECs. Unlike in the LECs from wild-type mice, TGFß2 failed to elicit an EMT response in LECs from RAGE knockout mice. The lack of RAGE also diminished TGFß2-mediated Smad signaling. In addition, treatment with TGFß2 increased IL-6 levels in LECs from wild-type mice but not in those from RAGE knockout mice. Treatment of human LECs with the RAGE inhibitor FPS-ZM1 reduced TGFß2-mediated Smad signaling and the EMT response. Unlike that in wild-type lenses, the removal of fiber cell tissue in RAGE knockout lenses did not result in elevated levels of α-smooth muscle actin (α-SMA), fibronectin (FN), and integrin ß1 in capsule-adherent LECs. Taken together, these results suggest that TGFß2 signaling is intricately linked to RAGE. Targeting RAGE could be explored as a therapeutic strategy against secondary cataracts.

Inhibitory effect of caffeic acid on advanced glycation end product-induced renal fibrosis in vitro: A potential therapeutic target.

Advanced glycation end products (AGEs) are formed from amino acids and reducing sugars through nonenzymatic Maillard reaction. AGEs are known to induce oxidative stress, which may cause fibrosis or cancer. In this study, we investigated the protective effect of caffeic acid (CA) on AGE-mediated kidney epithelial to mesenchymal transition (EMT) in human HK-2 cells. Exposure to 100 µg/mL of AGEs by kidney epithelial cells raised the production of reactive oxygen species by 5.2-fold and decreased levels of glutathione. In addition, cardamonin, a ß-catenin inhibitor, was used to determine the signaling pathway for ß-catenin in which cardamonin inhibited the AGEs-induced translocation of ß-catenin into the nucleus, resulting in an inhibition of the EMT process. Similarly, our findings showed that, close to the control level, CA treatment decreased AGE-mediated oxidative stress, loss of E-cadherin expression, and overexpression of α-smooth muscle actin and fibronectin by inactivation of the ß-catenin pathway. Furthermore, AGE treatment enhanced the expression of collagen type I (1.99-fold) as well as the activity of metalloproteinases 2 (1.86-fold) and 9 (2.79-fold), but such increase was inhibited by the pretreatment of CA. In conclusion, this study determined the inhibitory effect of CA on AGE-induced ß-catenin signaling, which prevented the occurrence of EMT in kidney epithelial cells. This suggests that CA may be a potential target for AGE-induced renal fibrosis. PRACTICAL APPLICATION: Exposure of kidney epithelial cells to advanced glycation end products (AGEs) leads to a rise in reactive oxygen species and a decrease in glutathione, thereby increasing oxidative stress that may cause fibrosis. However, treatment of kidney cells with caffeic acid (CA) prior to their exposure to AGEs lowers oxidative stress and decreases fibrosis. This research reveals the beneficial influence of CA on renal fibrosis in laboratory-cultured kidney cells (in vitro), which makes CA a potential therapeutic target for AGE-induced fibrosis.

Kynurenic Acid Protects Against Ischemia/Reperfusion-Induced Retinal Ganglion Cell Death in Mice.

BACKGROUND: Glaucoma is an optic neuropathy and involves the progressive degeneration of retinal ganglion cells (RGCs), which leads to blindness in patients. We investigated the role of the neuroprotective kynurenic acid (KYNA) in RGC death against retinal ischemia/reperfusion (I/R) injury. METHODS: We injected KYNA intravenously or intravitreally to mice. We generated a knockout mouse strain of kynurenine 3-monooxygenase (KMO), an enzyme in the kynurenine pathway that produces neurotoxic 3-hydroxykynurenine. To test the effect of mild hyperglycemia on RGC protection, we used streptozotocin (STZ) induced diabetic mice. Retinal I/R injury was induced by increasing intraocular pressure for 60 min followed by reperfusion and RGC numbers were counted in the retinal flat mounts. RESULTS: Intravenous or intravitreal administration of KYNA protected RGCs against I/R injury. The I/R injury caused a greater loss of RGCs in wild type than in KMO knockout mice. KMO knockout mice had mildly higher levels of fasting blood glucose than wild type mice. Diabetic mice showed significantly lower loss of RGCs when compared with non-diabetic mice subjected to I/R injury. CONCLUSION: Together, our study suggests that the absence of KMO protects RGCs against I/R injury, through mechanisms that likely involve higher levels of KYNA and glucose.

Aspirin inhibits TGFß2-induced epithelial to mesenchymal transition of lens epithelial cells: selective acetylation of K56 and K122 in histone H3.

Posterior capsule opacification (PCO) is a complication after cataract surgery that can disrupt vision. The epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) in response to transforming growth factor ß2 (TGFß2) has been considered an obligatory mechanism for PCO. In this study, we tested the efficacy of aspirin in inhibiting the TGFß2-mediated EMT of human LECs, LECs in human lens capsular bags, and lensectomized mice. In human LECs, the levels of the EMT markers α-smooth muscle actin (α-SMA) and fibronectin were drastically reduced by treatment with 2 mM aspirin. Aspirin also halted the EMT response of TGFß2 when introduced after EMT initiation. In human capsular bags, treatment with 2 mM aspirin significantly suppressed posterior capsule wrinkling and the expression α-SMA in capsule-adherent LECs. The inhibition of TGFß2-mediated EMT in human LECs was not dependent on Smad phosphorylation or MAPK and AKT-mediated signaling. We found that aspirin significantly increased the acetylation of K56 and K122 in histone H3 of human LECs. Chromatin immunoprecipitation assays using acetyl-H3K56 or acetyl-H3K122 antibody revealed that aspirin blocked the TGFß2-induced acetylation of H3K56 and H3K122 at the promoter regions of ACTA2 and COL1A1. After lensectomy in mice, we observed an increase in the proliferation and α-SMA expression of the capsule-adherent LECs, which was ameliorated by aspirin administration through drinking water. Taken together, our results showed that aspirin inhibits TGFß2-mediated EMT of LECs, possibly from epigenetic down-regulation of EMT-related genes.

Correction: Systemically administered peptain-1 inhibits retinal ganglion cell death in animal models: implications for neuroprotection in glaucoma.

[This corrects the article DOI: 10.1038/s41420-019-0194-2.].

Systemically administered peptain-1 inhibits retinal ganglion cell death in animal models: implications for neuroprotection in glaucoma.

Axonal degeneration and death of retinal ganglion cells (RGCs) are the primary causes of vision loss in glaucoma. In this study, we evaluated the efficacy of a peptide (peptain-1) that exhibits robust chaperone and anti-apoptotic activities against RGC loss in two rodent models and in cultured RGCs. In cultures of rat primary RGCs and in rat retinal explants peptain-1 significantly decreased hypoxia-induced RGC loss when compared to a scrambled peptide. Intraperitoneally (i.p.) injected peptain-1 (conjugated to a Cy7 fluorophore) was detected in the retina indicative of its ability to cross the blood-retinal barrier. Peptain-1 treatment inhibited RGC loss in the retina of mice subjected to ischemia/reperfusion (I/R) injury. A reduction in anterograde axonal transport was also ameliorated by peptain-1 treatment in the retina of I/R injured mice. Furthermore, i.p. injections of peptain-1 significantly reduced RGC death and axonal loss and partially restored retinal mitochondrial cytochrome c oxidase subunit 6b2 (COX 6b2) levels in rats subjected to five weeks of elevated intraocular pressure. We conclude that i.p. injected peptain-1 gains access to the retina and protects both RGC somas and axons against the injury caused by I/R and ocular hypertension. Based on these findings, peptain-1 has the potential to be developed as an efficacious neuroprotective agent for the treatment of glaucoma.

ERK/Nrf2 pathway activation by caffeic acid in HepG2 cells alleviates its hepatocellular damage caused by t-butylhydroperoxide-induced oxidative stress.

BACKGROUND: Several studies have found that caffeic acid (CA), a well-known phytochemical, displays important antioxidant and anti-cancer activities. However, no evidence exists on the protective effect and its mechanisms that CA treatment alone has against oxidative stress induced by tert-butyl hydroperoxide (t-BHP) in HepG2 cells. METHODS: Hepatoprotective activities such as cell viability, mRNA expression, and report gene assay were measured using HepG2 cell. Three types of genes and proteins related with detoxification in liver were used for measuring the hepatoprotective effects. Statistical analysis was performed using one-way ANOVA test and differences among groups were evaluated by Tukey's studentized range tests. RESULTS: The present study indicate that treatment with CA up-regulates heme oxygenase-1 (HO-1) and glutamate-cysteine ligase (GCL) mRNA and protein expressions in a CA-dose-dependent manner. In addition, translocation of nuclear factor-E2 p45-related factor (Nrf2) from the cytoplasm to the nucleus and phosphorylation of extracellular signal-regulated kinase, ERK and c-Jun N-terminal kinase, JNK which have been shown to be involved in mitogen-activated protein kinases, MAPKs are significantly enhanced by CA treatment. Furthermore, in cell nuclei, CA enhances the 5'-flanking regulatory region of human antioxidant response element (ARE) and activates the ARE binding site. CONCLUSION: Therefore, CA proved to be a stimulant of the expression of detoxification enzymes such as HO-1, GCLC, and GCLM through the ERK/Nrf2 pathway, and it may be an effective chemoprotective agent for protecting liver damage against oxidative damage.

Protective effects of chebulic acid from Terminalia chebula Retz. against t-BHP-induced oxidative stress by modulations of Nrf2 and its related enzymes in HepG2 cells.

Although chebulic acid isolated from Terminalia chebular has diverse biological effects, its effects on the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and the expression of downstream genes have not been elucidated. The purpose of this research is to investigate the hepatoprotective mechanism of chebulic acid against oxidative stress produced by tert-butyl hydroperoxide (t-BHP) in liver cells. The treatment with chebulic acid attenuated cell death in t-BHP-induced HepG2 liver cells and increased intracellular glutathione content, upregulated the activity of heme oxygenase-1, and also increased the translocation of Nrf2 into the nucleus and Nrf2 target gene expression in a dose-dependent manner. The exposure of chebulic acid activated the phosphorylation of mitogen-activated protein kinases. The overall result is that chebulic acid has cytoprotective effect on t-BHP-induced hepatotoxicity in HepG2 cells through Nrf2-mediated antioxidant enzymes.

Ensuring reproducibility and ethics in animal experiments reporting in Korea using the ARRIVE guideline.

The aim of this study is to evaluate the reporting quality of animal experiments in Korea using the Animals in Research: Reporting In Vivo Experiments (ARRIVE) guideline developed in 2010 to overcome the reproducibility problem and to encourage compliance with replacement, refinement and reduction of animals in research (3R's principle). We reviewed 50 papers published by a Korean research group from 2013 to 2016 and scored the conformity with the 20-items ARRIVE guideline. The median conformity score was 39.50%. For more precise evaluation, the 20 items were subdivided into 57 sub-items. Among the sub-items, status of experimental animals, housing and husbandry were described under the average level. Microenvironment sub-items, such as enrichment, bedding material, cage type, number of companions, scored under 10%. Although statistical methods used for the studies were given in most publications (84%), sample size calculation and statistical assumption were rarely described. Most publications mentioned the IACUC approval, but only 8% mentioned welfare-related assessments and interventions, and only 4% mentioned any implications of experimental methods or findings for 3R. We may recommend the revision of the present IACUC proposal to collect more detailed information and improving educational program for animal researchers according to the ARRIVE guideline.

Matrix-bound AGEs enhance TGFß2-mediated mesenchymal transition of lens epithelial cells via the noncanonical pathway: implications for secondary cataract formation.

Advanced glycation end products (AGEs) are post-translational modifications formed from the reaction of reactive carbonyl compounds with amino groups in proteins. Our laboratory has previously shown that AGEs in extracellular matrix (ECM) proteins promote TGFß2 (transforming growth factor-beta 2)-mediated epithelial-to-mesenchymal transition (EMT) of lens epithelial cells (LECs), which could play a role in fibrosis associated with posterior capsule opacification. We have also shown that αB-crystallin plays an important role in TGFß2-mediated EMT of LECs. Here, we investigated the signaling mechanisms by which ECM-AGEs enhance TGFß2-mediated EMT in LECs. We found that in LECs cultured on AGE-modified basement protein extract (AGE-BME), TGFß2 treatment up-regulated the mesenchymal markers α-SMA (α-smooth muscle actin) and αB-crystallin and down-regulated the epithelial marker E-cadherin more than LECs cultured on unmodified BME and treated with TGFß2. Using a Multiplex Assay, we found that AGE-BME significantly up-regulated the noncanonical pathway by promoting phosphorylation of ERK (extracellular signal-regulated kinases), AKT, and p38 MAPK (mitogen-activated protein kinases) during TGFß2-mediated EMT. This EMT response was strongly suppressed by inhibition of AKT and p38 MAPK phosphorylation. The AKT inhibitor LY294002 also suppressed TGFß2-induced up-regulation of nuclear Snail and reduced phosphorylation of GSK3ß. Inhibition of Snail expression suppressed TGFß2-mediated α-SMA expression. αB-Crystallin was up-regulated in an AKT-dependent manner during AGE-BME/TGFß2-mediated EMT in LECs. The absence of αB-crystallin in LECs suppressed TGFß2-induced GSK3ß phosphorylation, resulting in lower Snail levels. Taken together, these results show that ECM-AGEs enhance the TGFß2-mediated EMT response through activation of the AKT/Snail pathway, in which αB-crystallin plays an important role as a linker between the TGFß2 and AGE-mediated signaling pathways.

Protective effects of chebulic acid on alveolar epithelial damage induced by urban particulate matter.

BACKGROUND: Chebulic acid (CA) isolated from T. chebula, which has been reported for treating asthma, as a potent anti-oxidant resources. Exposure to ambient urban particulate matter (UPM) considered as a risk for cardiopulmonary vascular dysfunction. To investigate the protective effect of CA against UPM-mediated collapse of the pulmonary alveolar epithelial (PAE) cell (NCI-H441), barrier integrity parameters, and their elements were evaluated in PAE. METHODS: CA was acquired from the laboratory previous reports. UPM was obtained from the National Institutes of Standards and Technology, and these were collected in St. Louis, MO, over a 24-month period and used as a standard reference. To confirm the protection of PAE barrier integrity, paracellular permeability and the junctional molecules were estimated with determination of transepithelial electrical resistance, Western Blotting, RT-PCR, and fluorescent staining. RESULTS: UPM aggravated the generation of reactive oxygen species (ROS) in PAE and also decreased mRNA and protein levels of junction molecules and barrier integrity in NCI-H441. However, CA repressed the ROS in PAE, also improved barrier integrity by protecting the junctional parameters in NCI-H411. CONCLUSIONS: These data showed that CA resulted in decreased UPM-induced ROS formation, and the protected the integrity of the tight junctions against UPM exposure to PAE barrier.

Plantamajoside from Plantago asiatica modulates human umbilical vein endothelial cell dysfunction by glyceraldehyde-induced AGEs via MAPK/NF-κB.

BACKGROUND: Plantago asiatica has been traditionally used for traditional medicine around East Asia. Plantamajoside (PM), which is isolated from this plant, is known for biological properties including anti-inflammation and antioxidant activity. To demonstrate the biological activity of PM against endothelial dysfunction induced by advanced glycation end-products (AGEs), a cellular inflammatory mechanism system was evaluated in human umbilical vein endothelial cells (HUVECs). METHODS: We obtained PM through previous research in our laboratory. We formed the AGEs from bovine serum albumin with glyceraldehyde in the dark for seven days. To confirm the modulation of the inflammatory mechanism in endothelial dysfunction, we quantified the various pro-inflammatory cytokines and endothelial dysfunction-related proteins in the HUVECs with Western blotting and with real-time and quantitative real-time polymerase chain reactions. RESULTS: Co-treatment with PM and AGEs significantly suppressed inflammatory cytokines and adhesion molecule expression. Moreover, the PM treatment for down-regulated inflammatory signals and blocked monocyte adhesion on the HUVECs. CONCLUSIONS: Theses results demonstrated that PM, as a potential natural compound, protects AGE-induced endothelial cells against inflammatory cellular dysfunction.