Kwou-Yeung Wu

Endoplasmic reticulum stress implicated in the development of renal fibrosis.

Endoplasmic reticulum (ER) stress-associated apoptosis plays a role in organ remodeling after insult. The effect of ER stress on renal tubular damage and fibrosis remains controversial. This study aims to investigate whether ER stress is involved in tubular destruction and interstitial fibrosis in vivo. Renal cell apoptosis was proven by terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) stain and poly-ADP ribose polymerase expression in the unilateral ureteral obstruction (UUO) kidney. ER stress was evoked and confirmed by the upregulation of glucose-regulated protein 78 (GRP78) and the common Lys-Asp-Glu-Leu (KDEL) motif of ER retention proteins after UUO. ER stress-associated proapoptotic signals, including B-cell chronic lymphocytic leukemia (CLL)/lymphoma 2-associated × protein (BAX) expression, caspase-12 and c-Jun N-terminal kinase (JNK) phosphorylation, were activated in the UUO kidney. Prolonged ER stress attenuated both unsplicing and splicing X-box binding protein 1 (XBP-1) protein expression, but continued to activate inositol-requiring 1α (IRE1α)-JNK phosphorylation, protein kinase RNA-like endoplasmic reticulum kinase (PERK), eukaryotic translation initiation factor 2α subunit (eIF2α), activating transcription factor (ATF)-4, CCAAT/enhancer binding protein (C/EBP) homologous protein (CHOP) and cleavage activating transcription factor 6 (cATF6)-CHOP signals, which induce ER stress-related apoptosis but attenuate adaptive unfolded protein responses in UUO kidneys. However, renal apoptosis and fibrosis were attenuated in candesartan-treated UUO kidney. Candesartan was associated with maintenance of XBP-1 expression and attenuated ATF4, cATF6 and CHOP protein expression. Taken together, results show that overwhelming ER stress leads to renal cell apoptosis and subsequent fibrosis; and candesartan, at least in part, restores renal integrity by blocking ER stress-related apoptosis. Reducing ER stress may present a way to attenuate renal fibrosis.

Heat shock induces apoptosis through reactive oxygen species involving mitochondrial and death receptor pathways in corneal cells.

Although many studies have been performed to elucidate the molecular consequences of ultraviolet irradiation, little is known about the effect of infrared radiation on ocular disease. In addition to photons, heat is generated as a consequence of infrared irradiation, and heat shock is widely considered to be an environmental stressor. Here, we are the first to investigate the biological effect of heat shock on Statens Seruminstitut Rabbit Cornea (SIRC) cells. Our results indicate that heat shock exhibits effective cell proliferation inhibition by inducing apoptosis. Heat shock triggers the mitochondrial apoptotic pathway indicated by a change in Bax/Bcl-2 ratios, resulting in caspase-9 activity. In addition, heat shock triggered the death receptor apoptotic pathway indicated by a change in Fas ligand expression, resulting in caspase-8 activity. Furthermore, we also found that generation of reactive oxygen species (ROS) is a critical mediator in heat shock-induced apoptosis. In addition, the antioxidant vitamin C significantly decreased heat shock-mediated apoptosis. Taken together, these findings suggest a critical role for ROS involving mitochondrial and death receptor pathways in heat shock-mediated apoptosis of cornea cells.

Effect of Latanoprost on Cultured Porcine Corneal Stromal Cells

Purpose: Latanoprost reduces intraocular pressure mainly by enhancing uveoscleral outflow that may be involved in the decreased of extracellular matrixes such as collagens. However, the effect of latanoprost on corneal stromal cells is not well understood. In the current study, we investigated the changes of cultured porcine corneal stromal cells upon exposure to latanoprost. Methods: Porcine corneal stromal cells were acquired from primary culture and maintained in fetal bovine serum–containing medium. Cells were estimated on 3H-thymidine,3H-leucine,3H-uridine,3H-proline uptakes and migration. Dead and living cells were estimated with MTT assay. The changes of type 1 collagen and fibronectin proteins were detected by means of immunofluorescent staining and Western blot assay. Intracellular free Ca2 + ([Ca2 +]i) mobility was studied by spectrofluorophotometer after loading with fura-2-AM. Results: Latanoprost has remarkable effects inhibiting cultured corneal stromal cells on 3H-thymidine,3H-leucine,3H-uridine,3H-proline uptakes and cellular migration. The inhibitory effects are in a dose-dependent manner at concentrations ranging from 10− 5, 10− 6, 10− 7 to 10− 8 M. The 50% inhibitory dosages (ID50) for latanoprost to corneal stromal cells, as measured by 3H-thymidine uptake,3H-uridine uptake,3H-leucine uptake,3H-proline uptakes and cellular migration were 5.01 × 10− 6 M, 2.81 × 10− 6 M, 2.09 × 10− 6 M, 3.89 × 10− 7 M and 2.2 × 10− 6 M, respectively. In the presence of latanoprost, the cellular MTT values were also decreased significantly. Immunofluorescent staining displayed that latanoprost changed type 1 collagen distribution in cultured corneal stromal cells. Western blot assay revealed that latanoprost caused cells to decrease in fibronectin protein. In Ca2 +-containing buffer, latanoprost induced a significant rise in [Ca2 +]i at 10− 5 and 10− 6 M. Conclusions: These results indicate that latanoprost may induce the morphological and biochemical changes in cultured corneal stromal cells. Long-term use of latanoprost needs to be carefully monitored for change in corneal stroma.

CELLULAR CYTOTOXICITY OF ANTIGLAUCOMA DRUGS IN CULTURED CORNEAL ENDOTHELIAL CELLS

In this study, the various antiglaucoma drugs including betaxolol, timolol, levobunolol, carteolol, brimonidine, dipivefrin, dorzolamide, brinzolamide, latanoprost, unoprostone, and pilocarpine were used to investigate the effects of cellular cytotoxicity in cultured bovine corneal endothelial cells. After exposure to the drugs in three dilutions, 1/100, 1/1,000, and 1/10,000, for 100 minutes, cells were estimated based on the release assay of lactate dehydrogenase (LDH) enzyme. It was found that cellular LDH was significantly released in the medium only at 1/100th dilution of betaxolol, brimonidine, dorzolamide, dipivefrin, latanoprost and unoprostone to 130%, 123%, 145%, 157%, 128% and 237%, respectively, compared with controls upon exposure to drugs for 100 minutes. Moreover, benzalkonium chloride preservative at the concentrations ranging from 0.001 to 0.00001mg/mL did not affect cellular LDH release in bovine corneal endothelial cells. These results indicate that high concentrations of antiglaucoma drugs may induce cytotoxicity in corneal endothelial cells.

Toxic Effects of Mitomycin-C on Cultured Ciliary Process Cells and Trabecular Meshwork Cells

Mitomycin-C has recently become an adjunct medication for inhibition of fibroblast proliferation in glaucoma filtering procedures. Prolonged postoperative ocular hypotony has been a frequent complication of trabeculectomy with mitomycin-C. In order to characterize the hypotony mechanism, we compared the toxic effects of mitomycin-C on cultured rabbit ciliary process cells and trabecular meshwork cells. The results indicate that mitomycin-C has a more marked effect on ciliary process cells on 3H-thymidine uptake than on trabecular meshwork cells at concentrations ranging from 10(-1) to 10(-5) mg/ml after 3-, 5- and 60-min treatment, respectively. The living cells after mitomycin-C treatment were estimated with MTT assay that was converted tetrazolium dye of living cells only into insoluble purple formazan crystals within mitochondria. In the presence of mitomycin-C for 3, 5, and 60 min, the cellular MTT values in ciliary process cells were more decreased than in trabecular meshwork cells. Depolarization of the trabecular meshwork cells with 50 mM KCl led to an increase in intracellular calcium concentration, whereas application of mitomycin-C at 10(-3) mg/ml resulted in decrease of KCl-induced intracellular calcium increase. Mitomycin-C (10(-3) mg/ml) decreased cAMP concentration in ciliary process cells following 3- and 5-min treatment; however, it did not significantly affect the cellular cAMP concentration after only a 1-min exposure. Mitomycin-induced marked ladder pattern of DNA fragmentation was observed in ciliary process tissues after treatment with 10(-1) mg/ml of mitomycin-C for 3 and 5 min. However, the DNA pattern in trabecular meshwork tissues was not obviously affected by mitomycin-C. These findings from our results indicate that mitomycin-induced ocular hypotony may result from damage to both ciliary process and trabecular meshwork tissues.

Impact of body mass on outcomes of geriatric postoperative acute kidney injury patients.

ABSTRACT Background: Acute kidney injury (AKI) frequently occurs in hospitalized patients, particularly in the elderly. However, studies on outcome-modifying factors in geriatric patients with AKI are absent, especially the influence of body mass index (BMI). Methods: We performed a retrospective analysis of a prospectively collected multicenter observational cohort, which enrolled elderly (≥65 years) who developed AKI after major surgery in the intensive care units. We analyzed in-hospital mortality within BMI category utilizing Cox proportional hazard regression analysis and generalized additive modeling. Results: Data of a total of 2,015 postoperative elderly patients were retrieved and analyzed. Generalized additive modeling showed that elderly AKI patients with a BMI between 21 and 31 kg/m2 (“normal”) had a lower mortality risk than those with a BMI of less than 21 kg/m2 (“underweight”) or 31 kg/m2 or greater (“obese”). Both “underweight” and “obese” individuals had a greater risk of mortality compared with patients with “normal” BMI. Conclusions: The U-shaped association of BMI with hospital mortality in geriatric AKI patients contains a widened base and a shifted nadir comparing with chronic dialysis and other AKI patients. This finding is interesting and warrants our attention.

The oculocardiac reflex during strabismus surgery: its relationship to preoperative clinical eye findings and subsequent postoperative emesis.

PURPOSE To compare the occurrence of significant bradycardia due to the oculocardiac reflex (OCR) during strabismus surgery and its relationship to preoperative clinical eye findings and subsequent development of postoperative emesis. METHODS The medical records of consecutive patients who underwent strabismus surgery August 2006 to June 2009 were retrospectively reviewed. OCR was defined as presence of dysrhythmia or a sudden heart rate decrease ≥ 20% after traction on the extraocular muscle. OCR incidence was compared between the first, second, and third (if any) extraocular muscles in patients who had multiple-muscle strabismus surgery and also between specific muscles (eg, medial rectus vs lateral rectus muscle). Associations with OCR were compared for different strabismus types. Vomiting was considered postoperative if it occurred before discharge of the patient at noon on the day following surgery. Risk factors for OCR and postoperative vomiting were evaluated by logistic regression analysis. RESULTS A total of 111 records were reviewed; 41 patients (37%) experienced OCR. Incidence of OCR and absence of OCR during traction of the first muscle were significantly associated with events during traction of the second (χ(2) = 36.681, P < 0.001) and third muscles (Fisher exact test, P = 0.030). The best predictors of OCR were the absence of fine stereopsis and a larger number of surgically treated muscles. Of the 111 patients, 30 (27%) who had postoperative vomiting, the best predictors were female sex and young age. CONCLUSIONS In our cohort, patients experiencing intraoperative OCR when the first extraocular muscle was manipulated during multiple-muscle strabismus surgery were likely to experience it again during traction of additional muscles. More severe postoperative vomiting was common in these patients. OCR was associated with the preoperative absence of fine stereopsis.

Pleiotropic role of atorvastatin in regulation of human retinal pigment epithelial cell behaviors in vitro

Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, possess pleiotropic effects that have been extended to modulation of various cellular behaviors. This study aimed at examining whether atorvastatin (AVN) modulates cell growth, adhesion, migration, and contraction of cultured human retinal pigment epithelium (RPE) cells. The in vitro effects of AVN on human RPE cells was analyzed in terms of cell proliferation, cell cycle, cell adhesion, migration, and contraction assays. The modulatory effect of AVN on TGF-β2-triggered signaling was determined by Western blotting detection. AVN at submicromolar dose exhibited no prominent morphological alteration and cytotoxicity, whereas it elicited cytostatic effect at concentrations higher than 1 μM. Cell cycle analysis showed that AVN induced growth arrest in both G1 and G2/M phases. AVN at 1 μM or higher concentrations significantly suppressed RPE cell adhesion. Cell migration and 3D collagen contraction assays showed that AVN significantly suppressed RPE cell migration and contractility, respectively. Mechanistically, AVN treatment transiently up-regulated phosphorylation of Akt, ERK1/2, and p38 MAPK, whereas down-regulated that of JNK1. Intriguingly, AVN pretreatment prominently attenuated the TGF-β(2)-mediated non-Smad signaling, including Akt, ERK1/2, p38 MAPK, and JNK1 phosphorylation. Besides, it directly reduced constitutive level of myosin regulatory light chain peptide MYL9 and mitigated the TGF-β(2)-induced phosphorylation of myosin phosphatase-targeting subunit 1, MYPT1. These in vitro findings strongly suggest that AVN possesses pleiotropic function on RPE cells, including anti-proliferation, anti-adhesion, anti-migration as well as anti-contraction. In conclusion, AVN treatment may be considered a useful therapy for proliferative vitreoretinal diseases.

Effects of Antiglaucoma Drugs on Calcium Mobility in Cultured Corneal Endothelial Cells

The aim of this study was to estimate the effects of various antiglaucoma drugs including betaxolol, timolol, levobunolol, brimonidine, carteolol, dipivefrin, dorzolamide, brinzolamide, latanoprost, unoprostone, and pilocarpine on intracellular free Ca2+ ([Ca2+]i) mobility in cultured bovine corneal endothelial cells. Various antiglaucoma drugs were diluted from original concentrations to 1/100, 1/1,000, and 1/10,000. The [Ca2+]i mobility was studied by spectrofluorophotometry after loading with the ester of fura‐2 (fura‐2/AM). It was found that timolol (58 μM and 5.8 μM), levobunolol (171 μM, 17.1 μM, and 1.71 μM), betaxolol (162 μM, 16.2 μM, and 1.62 μM), carteolol (680 μM and 68 μM), dipivefrin (28 μM and 2.8 μM), dorzolamide (616 μM and 61.6 μM), brinzolamide (260 μM), latanoprost (1.1 μM), unoprostone (28.2 μM, 2.82 μM, and 0.282 μM), and pilocarpine (408 μM and 40.8 μM) induced a significant increase in [Ca2+]i. Nevertheless, only brimonidine (68 μM and 6.8 μM) decreased [Ca2+]i concentration significantly. Benzalkonium chloride preservative did not affect [Ca2+]i after addition of 0.001, 0.0001 and 0.00001 mg/mL to cells. These results indicate that all antiglaucoma drugs may affect the physiologic function of corneal endothelial cells through change of [Ca2+]i.mobility.

Inhibition of endothelin-1 and KCL-induced increase of [CA2+]i by antiglaucoma drugs in cultured A7r5 vascular smooth-muscle cells.

Over contraction of vascular smooth muscle may result in ischemia to ocular neuronal cells and deteriorate the glaucoma. The purpose of this study was to investigate the inhibitory effects of various commercial antiglaucoma drugs including brimonidine, dipivefrin, betaxolol, timolol, levobunolol, carteolol, brinzolamide, dorzolamide, unoprostone, latanoprost, pilocarpine, and preservative benzalkonium chloride on endothelin-1(ET-1) and KCl-induced increase of intracellular free Ca2+ ([Ca2+]i) in cultured rat A7r5 vascular smooth muscle cells. These drugs were diluted from original concentrations to 1/100, 1/1000, and 1/10000. [Ca2+]i mobility was analyzed by spectrofluorometry after loading with fura-2-AM. Betaxolol, timolol, levobunolol, and carteolol were found to inhibit KCl-induced release of [Ca2+]i in a dose-dependent manner. High concentrations of betaxolol, timolol, levobunolol, carteolol, and unoprostone also inhibited ET-1-induced increase of [Ca2+]i in A7r5 cells. However, ET-1- and KCl-induced increase of [Ca2+]i was not diminished by other drugs including brimonidine, dipivefrin, brinzolamide, dorzolamide, latanoprost, pilocarpine, and benzalkonium chloride. These results indicate that high concentrations of unoprostone and beta-adrenergic blocking agents including betaxolol, timolol, levobunolol, and carteolol may inhibit ET-1-induced increase of [Ca2+]i. The mechanism may be mediated by inhibition of extracellular calcium influx via blocking of L-type voltage-dependent Ca2+ channel in A7r5 cells.