fan Yang

Inhibition of Pirfenidone on TGF-beta2 Induced Proliferation, Migration and Epithlial-Mesenchymal Transition of Human Lens Epithelial Cells Line SRA01/04

Background Posterior capsular opacification (PCO) is a common complication of cataract surgery. Transforming growth factor-β2 (TGF-β2) plays important roles in the development of PCO. The existing pharmacological treatments are not satisfactory and can have toxic side effects. Methodologies/Principal Findings We evaluated the effect of pirfenidone on proliferation, migration and epithlial-mesenchymal transition of human lens epithelial cell line SRA01/04 (HLECs) in vitro. After treatment with 0, 0.25, and 0.5 mg/ml pirfenidone, cell proliferation was measured by MTT assay. Cell viability was determined by trypan blue exclusion assay and measurement of lactate dehydrogenase (LDH) activity released from the damaged cells. And cell migration was measured by scratch assay in the absence or presence of transforming growth factor-β2 (TGF-β2). The expressions of TGF-β2 and SMADs were evaluated with real-time RT-PCR, western blot, and immunofluorescence analyses. The mesenchymal phenotypic marker fibronectin (FN) was demonstrated by Immunocytofluorescence analyses. The cells had high viability, which did not vary across different concentrations of pirfenidone (0 [control] 0.3, 0.5 or 1.0 mg/ml) after 24 hours. Pirfenidone (0∼0.5 mg/ml) had no significant cytotoxicity effect on SRA01/04 by LDH assay. Pirfenidone significantly inhibited the proliferation and TGF-β2-induced cell migration and the effects were dose-dependent, and inhibited TGF-β2-induced fibroblastic phenotypes and TGF-β2-induced expression of FN in SRA01/04 cells. The cells showed dose-dependent decreases in mRNA and protein levels of TGF-β2 and SMADs. Pirfenidone also depressed the TGF-β2-induced expression of SMADs and blocked the nuclear translocation of SMADs in cells. Conclusion Pirfenidone inhibits TGF-β2-induced proliferation, migration and epithlial-mesenchymal transition of human lens epithelial cells line SRA01/04 at nontoxic concentrations. This effect may be achieved by down regulation of TGF-β/SAMD signaling in SRA01/04 cells.

Cytochrome Oxidase 2D6 Gene Polymorphism in Primary Open-Angle Glaucoma With Various Effects to Ophthalmic Timolol

AIMS Timolol is used topically for the treatment of glaucoma and metabolized by cytochrome P450 (CYP) 2D6 in the liver. The aim of this study is to test the hypothesis that CYP 2D6 single-nucleotide polymorphism (SNP) is associated with drug effects of ophthalmic timolol. METHODS A total of 133 primary open-angle glaucoma (POAG) subjects underwent the ophthalmic single timolol administration and the drug effects were observed, including lowering the effects of intraocular pressure (IOP) and side effects (i.e., appearing bradycardia). Eight SNPs of CYP2D6 were investigated in 73 subjects by a SNPstream genotyping system. The relationship between the effects of timolol and CYP2D6 Arg296Cys and Ser486Thr genotype distribution in these POAG subjects was analyzed. RESULTS Topical timolol administration had significant effect on IOP (P = 0.000) and heart rate (HR) (P = 0.000) in all 133 subjects, and individual ocular hypotensive effect of timolol varied between 0 and 23 mmHg. Individual effect of HR varied between -31 and 10 beats per minute, in the present study. According to SNP genotyping in 73 subjects, there was no significant difference of IOP between subjects with different CYP2D6 Arg296Cys (P = 0.308) or Ser486Thr genotypes (P = 0.741). The effect of timolol on HR was significantly different between subjects with different Arg296Cys genotypes (P = 0.046). Timolol-induced bradycardia tended to occur in subjects with Arg296Cys CT and TT genotype when compared with CC genotype (P = 0.009). CONCLUSIONS CYP2D6 SNP Arg296Cys appeared to be correlative with the intersubject variability seen with timolol in POAG subjects. Subjects with CC genotype trended to avoid timolol-induced bradycardia, and subjects with TT genotype trended to have poorer timolol-induced ocular hypotensive effects.

Experimental studies on soft contact lenses for controlled ocular delivery of pirfinedone: in vitro and in vivo

Abstract Context: Pirfinedone (PFD) is a novel agent which has the potential to prevent scarring in the eyes. The 0.5% PFD eye drops exhibits poor bioavailability. Whereas, the feasibility of using contact lens as ocular drug delivery device initiated novel possibilities. Objective: To evaluate the delivery of PFD by soft contact lens (SCL) in vivo, we screened the most suitable lens material for PFD among various commercially available SCL materials in vitro. Material and methods: Firstly, 11 different SCLs (−1.00 diopter) were respectively soaked in 2 ml of 0.05% PFD-loading solution for 24 h to fully absorb drug, and then placed in fresh phosphate buffered saline (PBS) to release the drug. PFD concentration in PBS was determined by ultraviolet absorbance at 310 nm. Secondly, by immersing in 2 ml of 0.5% PFD eye drops for 24 h, the polymacon lens (0.00 diopter) was then placed on the cornea of New Zealand rabbits. PFD concentrations were detected by high performance liquid chromatography (HPLC) in tears, aqueous humor, conjunctiva, cornea, and sclera at different time points. Results: PFD showed some affinity for pHEMA-based lenses and the polymacon lens more slowly released more amount of PFD than any other lens in vitro (p < 0.001). Compared with eye drops, drug-loaded SCLs greatly enhanced the retention time and concentrations of PFD in cornea and aqueous humor and consequently improved the bioavailability of PFD. Conclusion: Polymacon-based SCL is probably a promising vehicle to be an effective ophthalmic delivery system for PFD.

Association of CYP2D6 Single-Nucleotide Polymorphism with Response to Ophthalmic Timolol in Primary Open-Angle Glaucoma—A Pilot Study

OBJECTIVES We aimed to investigate whether CYP2D6 polymorphisms were associated with the intraocular pressure (IOP)-lowering effect and systemic complications (especially bradycardia) of ophthalmic timolol. METHODS One hundred twenty-three primary open-angle glaucoma subjects (123 eyes) were treated with 0.5% aqueous formulations of ophthalmic timolol. IOP and heart rate were measured before and after timolol administration. DNA was extracted from venous leukocytes of all the subjects. Two single-nucleotide polymorphisms (SNPs) of CYP2D6, for example, rs16947 (2850C>T, R296C) and rs1135840 (4180C>G, S486T), were detected by restriction fragment length polymorphism analysis. RESULTS Neither rs16947 (P = 0.339) nor rs1135840 (P = 0.903) genotype was statistically correlated with the IOP-lowering effect of timolol eye drops. The rs16947 genotype was significantly associated with occurrence of bradycardia in primary open-angle glaucoma patients (P = 0.021). The patients with rs16947 CT (P = 0.043) or TT (P = 0.043) were more inclined to bradycardia than those with rs16947 CC, although there was no significant difference between CT and TT (P = 0.177). The analysis of variance showed no significant difference in heart rate (P = 0.559) among GG, GC, and CC groups. CONCLUSIONS CYP2D6 SNP rs16947 may confer susceptibility to timolol-induced bradycardia. Patients with CC genotype were unlikely to suffer from timolol-induced bradycardia, whereas those with TT genotype were found to suffer from timolol-induced bradycardia.

Down-regulation of 14-3-3 Zeta Inhibits TGF-β1–Induced Actomyosin Contraction in Human Trabecular Meshwork Cells Through RhoA Signaling Pathway

PURPOSE The aim of this study was to describe the expression and distribution of 14-3-3 zeta in trabecular meshwork (TM) cells and its regulatory role in the actomyosin system. METHODS The expression of 14-3-3 zeta was detected using Western blot analysis, RT-PCR, and immunofluorescence staining. TGF-β1 was used to induce cell contraction. Changes in the levels of 14-3-3 zeta, total RhoA, and the phosphorylation of myosin light chain (MLC) and cofilin were determined using Western blot analysis. The effects of 14-3-3 zeta knockdown on the actin cytoskeleton and focal adhesion were determined using immunofluorescence. The mRNA levels of fibronectin and collagen I and III were examined using quantitative RT-PCR. The contraction of TM cells was detected using collagen gel contraction (CGC) assays. The activation of the RhoA pathway was analyzed using a specific kit. RESULTS The 14-3-3 zeta protein was highly expressed in TM cells. Down-regulation of 14-3-3 zeta resulted in the following: a decrease in the phosphorylation of both MLC and cofilin, a decrease in the formation of stress fibers and focal adhesion, alteration of the mRNA composition of the extracellular matrix (ECM), and the inhibition of TGF-β1-induced cell contraction. In addition, silencing of 14-3-3 zeta directly decreased total RhoA levels in TM cells. CONCLUSIONS Collectively, our data suggest that 14-3-3 zeta plays a crucial role in regulating cytoskeletal structures, ECM homeostasis, and TGF-β1-induced contraction in TM cells by acting through the RhoA signaling pathway.

Modified Team-Based Learning in an Ophthalmology Clerkship in China.

Objective Team-based learning (TBL) is an increasingly popular teaching method in medical education. However, TBL hasn’t been well-studied in the ophthalmology clerkship context. This study was to examine the impact of modified TBL in such context and to assess the student evaluations of TBL. Methods Ninety-nine students of an 8-year clinical medicine program from Zhongshan Ophthalmic Centre, Sun Yat-sen University, were randomly divided into four sequential units and assigned to six teams with the same faculty. The one-week ophthalmology clerkship module included traditional lectures, gross anatomy and a TBL module. The effects of the TBL module on student performance were measured by the Individual Readiness Assurance Test (IRAT), the Group Readiness Assurance Test (GRAT), the Group Application Problem (GAP) and final examination scores (FESs). Students’ evaluations of TBL were measured by a 16-item questionnaire. IRAT and GRAT scores were compared using a paired t-test. One-way analysis of variance (ANOVA) and subgroup analysis compared the effects among quartiles that were stratified by the Basic Ophthalmology Levels (BOLs). The BOLs were evaluated before the ophthalmology clerkship. Results In TBL classes, the GRAT scores were significantly higher than the IRAT scores in both the full example and the BOL-stratified groups. It highlighted the advantages of TBL compared to the individual learning. Quartile-stratified ANOVA comparisons showed significant differences at FES scores (P < 0.01). In terms to IRAT, GRAT and GAP scores, there was no significant result. Moreover, IRAT scores only significantly differed between the first and fourth groups. The FES scores of the first three groups are significantly higher than the fourth group. Gender-specific differences were significant in FES but not the IRAT. Overall, 57.65% of student respondents agreed that TBL was helpful. Male students tended to rate TBL higher than female students. Conclusion The application of modified TBL to the ophthalmology clerkship curriculum improved students’ performance and increased students’ engagement and satisfaction. TBL should be further optimized and developed to enhance the educational outcomes among multi-BOLs medical students.

Tetramethylpyrazine Attenuates Transdifferentiation of TGF-β2–Treated Human Tenon's Fibroblasts

PURPOSE To investigate the pharmacologic effect of tetramethylpyrazine (TMP) on human Tenons fibroblasts (HTFs), the cells implicated in scarring after filtration surgery. METHODS Transforming growth factor-β2 (TGF-β2) was used to stimulate a fibrotic phenotype in primary HTFs, and the influence of TMP on the fibrotic phenotype was assessed. Cell proliferation and cell cycle regulation were profiled. Immunofluorescence staining tracked proliferating cell nuclear antigen (PCNA) expression. Transwell assays monitored cell migration. Flow cytometry measured TMP toxicity. In addition, in TGF-β2-treated HTFs, Western blot and immunofluorescence were employed to assess the expression of α-smooth muscle actin (α-SMA). The TMP-mediated activity on cytoskeletal arrangements and extracellular matrix (ECM) accumulation in HTFs was evaluated using actin polymerization and Western blot assays. Moreover, TGF-β-dependent activation of Smad3 and p38 was examined by Western blot analysis. RESULTS In TGF-β2-treated HTFs, TMP reduced proliferation and migration but did not induce apoptosis. Moreover, TMP attenuated expression of α-SMA and suppressed stress fiber formation stimulated by profibrotic cytokine; it also counteracted TGF-β2-induced cytoskeletal rearrangements, morphologic changes, and ECM accumulation. Smad3 and p38 mitogen-activated protein kinase (MAPK) signaling were downstream of the TMP-sensitive effect. CONCLUSIONS Tetramethylpyrazine counteracts TGF-β2-mediated myofibroblast transdifferentiation and attenuates ECM component deposition and cell proliferation in HTFs, implicating TMP as a potential antifibrosis agent in glaucoma filtration surgery.

Comparison of Efficacy Between Endoscopic Cyclophotocoagulation and Alternative Surgeries in Refractory Glaucoma: A Meta-analysis

AbstractRefractory glaucoma refers to uncontrolled intraocular pressure (IOP) despite anti-glaucoma medication and surgical treatment, which remains a challenge to be treated. The objective of this study is to evaluate and statistically compare the clinical efficacy between endoscopic cyclophotocoagulation (ECP) and alternative surgical techniques in the treatment of refractory glaucoma in this article, as a meta-analysis.Data sources are China Biomedical Database (Sinomed, online version), China National Knowledge Infrastructure (CNKI), Cqvip, Wanfang database, and PubMed.The randomized controlled trial (RCT) and case–control study literatures evaluating the clinical efficacy between ECP and other surgical techniques were searched electronically from public databases. The methodology quality of the retrieved articles was evaluated according to the RCT or case–control study criteria. The success rate of treatment, intraocular pressure (IOP) and visual acuity were statistically compared. RevMan 5.3 software was used for the meta-analysis.In total, 6 relevant control studies were selected in this study with a total sampling of 429 cases (429 eyes), including 204 eyes in the ECP group and 225 in the non-ECP group. Meta-analysis demonstrated that the clinical efficacy did not significantly differ between 2 groups (P > 0.05). Postoperative IOP was dramatically reduced in both groups. However, it was difficult to evaluate the combined influence of ECP and non-ECP therapies upon IOP reduction.In conclusion, ECP and non-ECP treatment yielded almost equivalent clinical efficacy in treating refractory glaucoma. The IOP-lowering degree, safety, and incidence of complications remain to be further elucidated by RCTs with a larger sample size.

Phakomatosis Pigmentovascularis Associated With Sturge-Weber Syndrome, Ota Nevus, and Congenital Glaucoma.

AbstractPhakomatosis pigmentovascularis (PPV) is a rare congenital malformation syndrome that is characterized by a combination of capillary abnormalities and dermal melanocytosis.We describe 3 cases of PPV combined with bilateral Sturge–Weber syndrome (SWS), Ota nevus, and congenital glaucoma.Case 1 was a 2-year-old boy. Facial port-wine stains distributed along the 3 branches of his trigeminal nerves, which suggested the existence of SWS. Gray-blue patches were spread over the frontal and temporal areas of bilateral face, waist, buttocks, and thigh. Bilateral triangular alopecia was found on the temporal scalp. The diagnosis of Ota nevus was made by the bilateral scleral malanocystosis. Increased intraocular pressure, enlarged cornea, and pathologic optic disc cupping supported the diagnoses of infantile bilateral glaucoma. Case 2 was a 4-year-old boy. Port-wine stains were found on the face along the 3 branches of the trigeminal nerve and distributed along the trunk, arms, and legs. Mongolian spots spread over his frontal and temporal areas of the bilateral face, waist, buttocks, thigh, abdomen, and back. Infantile glaucoma was found in both eyes. Ota nevus were found in the both eyes. Optic coherent tomography (OCT) scans revealed increased thickness of choroid. Case 3 was a 5-year-old boy. Besides Ota nevus and infantile glaucoma in both eyes, color Doppler ultrasonography showed choroidal hemagioma. OCT scan showed increased choroidal thickness. The bilateral triangular alopecia on the childs temporal scalp was similar to that of Case 1. Cases 1 and 2 presented with port-wine stain patches that were consistent with the characteristic manifestation of PPV type IIb. However, the CMTC of Case 3 met the diagnostic criteria for PPV type Vb.Case 1 was treated with trabeculotomies in both eyes. For Cases 2 and 3, surgical interventions were not considered due to the high risks of antiglaucomatous operation complications. We prescribed them antiglaucoma indications.The simultaneously coexistence of PPV with SWS, Ota nevus, and congenital glaucoma is rare. In the clinic, additional detailed examinations and tests of PPV patients to exclude other ocular abnormalities or extraocular involvements are necessary.

Preparation and evaluation of HPMC-based pirfenidone solution in vivo

Abstract Context: Pirfenidone (PFD) has exhibited therapeutic potential in the treatment of cell proliferative disorders. The previously developed 0.5% water-based PFD eye drops by our team exhibited antiscarring effectiveness and ocular safety but with a limit of short half-life and poor bioavailability. Objective: To increase bioavailability of the water-based PFD eye drops, we prepared a viscous solution by adding hydroxypropyl methylcellulose (HPMC, F4M), which acted as a viscosity-enhancer. Subsequently, we compared the HPMC-based PFD solution with the water-based PFD eye drops. Materials and methods: PFD solution with 1% HPMC (w/v) was prepared, and the viscosities at different shear rates were measured to investigate its rheology. PFD concentrations in the tear, aqueous humor, conjunctiva, cornea, and sclerae of New Zealand rabbits were detected at different time points with high-performance liquid chromatography (HPLC) following single instillation of the 0.5% PFD (w/v) water-based eye drops or HPMC-based solution. Results: Compared with the 0.5% water-based PFD eye drops, the HPMC-based solution increased the PFD levels in tears and prolonged the residence time from 10 to more than 20 min (p < .01). Consequently, the concentrations of PFD in aqueous humor, conjunctiva, cornea, and sclera were elevated to varying degrees until 90 min after topical administration. Conclusions: The developed formulation possesses a same readily administration and simple preparation as the PFD eye drops; however, the HPMC-based solution exhibited the higher bioavailability.