Nicotinamide mononucleotide increases cell viability and restores tight junctions in high-glucose-treated human corneal epithelial cells via the SIRT1/Nrf2/HO-1 pathway.

BACKGROUND: Diabetes mellitus (DM)-related corneal epithelial dysfunction is a severe ocular disorder; however, the effects of nicotinamide mononucleotide (NMN) on high-glucose (HG)-treated human corneal epithelial cells (HCECs) remain unclear. METHODS: We conducted an in-vitro study to examine the effects of NMN treatment on HG-treated HCECs. Cell viability was measured using trypan blue stain, mitochondrial membrane potential was measured using JC-1 stain, and intracellular reactive oxygen species and apoptosis assays were conducted using flow cytometry. Transepithelial electrical resistance (TEER) and zonula occludens-1 (ZO-1) immunofluorescence for tight junction examinations were conducted. Immunoblot analyses were conducted to analyze the expression of silent information regulator-1 (SIRT1), nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) of the SIRT1/Nrf2/HO-1 pathway. RESULTS: NMN increased cell viability by reducing cell damage, reducing apoptosis, increasing cell migration, and restoring tight junctions in HG-treated HCECs. By analyzing the expressions of SIRT1, Nrf2, HO-1, NMN demonstrated protective effects via the SIRT1/Nrf2/HO-1 pathway. CONCLUSIONS: NMN increases cell viability by reversing cell damage, reducing apoptosis, increasing cell migration, and restoring tight junctions in HG-treated HCECs, and these effects may be mediated by the SIRT1/Nrf2/HO-1 pathway.

Imaging and anatomical parameters of the lacrimal punctum and vertical canaliculus using optical coherence tomography.

Purpose: The anatomical parameters of normal lacrimal puncta and vertical canaliculus using optical coherence tomography (OCT) and the OCT imaging features of punctal lesions were analyzed to provide a basis for clinical diagnosis and treatment. Methods: From June to September 2019, 40 volunteers (80 eyes) from Tongji Hospital were enrolled. The external punctal diameter (ELP) was measured using slit-lamp microscopy and OCT. The internal lacrimal punctal diameter (ILP) at 100 µm, vertical canalicular length (VCL), and tear meniscus depth were measured by OCT with open eyes. Twenty-eight volunteers (56 eyes) underwent the same examinations with their eyes closed. The OCT imaging features of 26 patients (27 eyes) with lacrimal lesions were examined. Results: The ELP of the right and left healthy eyes under slit-lamp microscopy were 564.40 and 555.40 µm respectively. Under OCT, the ELP, ILP, and VCL of the right and left eyes were 628.20 um and 616.85 µm, 343.40 µm and 346.95 µm, 731.95 um and 709.20 µm respectively. The ELP was larger when measured by OCT than slit-lamp microscopy (p<0.05). Twenty-eight volunteers (56 eyes) had measurements taken under different conditions. The ELP, ILP, and VCL of the open and closed right eyes were 667.54 and 567.21 µm, 369.18 and 303.18 µm, 715.00 and 417.14 µm, respectively. The ELP, ILP, and VCL of the open and closed left eyes were 655.86 um and 551.68 µm, 369.25 um and 313.54 µm, 719.96 um and 433.89 µm respectively. The anatomical parameters of the open eyes were greater than those of the closed eyes (p<0.05). Thus, we identified the imaging features of lacrimal stenosis, punctal obstruction, punctal tear, lacrimal atresia, and lacrimal mass using OCT. Conclusions: OCT can be used to measure the anatomical parameters of lacrimal puncta and vertical canaliculus in vivo. In addition, OCT can detect punctal lesions in vivo and provide an objective basis for the clinical diagnosis and treatment of punctal lesions.

Microbiological contamination in donor corneas preserved for medium-term.

To evaluate the characteristics of microbiological contamination in donor corneas preserved for medium-term. A total of 82 donated corneas from June 1, 2014 to November 30, 2014 were retrospectively analyzed. The corneas were preserved in cornea chambers medium-term solution at 4-8 °C for keratoplasty. After removal of the central corneas for transplantation, the corneoscleral rims were put back into the medium for 1 month at room temperature (20-25 °C). The suspicious contaminated storage solutions indicated with transparency or color change were examined with bacteria and fungi cultivation for strain identification. The data collected included gender, age, procurement site and causes of death of donors, and follow-up of recipients. Statistical analysis was performed using Microsoft Excel and SPSS 24.0. Significance level was set at a P value < 0.05. The overall pathogen positive rate was 9.8% (n = 8), including 7 (87.5%) fungi and 1 (12.5%) bacteria. They were 2 (2.44%) Fusarium, 2 (2.44%) Chromomycosis, 1 (1.22%) Candida albicans, 1 (1.22%) Aspergillus versicolor, 1 (1.22%) Acremonium species, and 1 (1.22%) Enterococcus. 5 contaminated corneas were used for penetrating keratoplasty; although four out of five (80%) had not been given antifungal drugs during more than 6 months following-up period, none of the recipients was infected with a graft. Donor age (P = 0.839), gender (P = 0.062), procurement sites (P = 0.713) and cause of death (P = 0.711) had no statistically significant influence on the contamination rate. All donor corneas have a possibility of microbiological contamination. Strict tissue preservation protocol but not antifungal drugs following keratoplasty seems necessary to prevent graft infection.

A New Isolation Method of Human Lacrimal Canaliculus Epithelial Stem Cells by Maintaining Close Association with Their Niche Cells.

Purpose: To investigate whether lacrimal canaliculus epithelial stem cells (LCESC) could be isolated and expanded in vitro. Methods: The lacrimal canaliculus epithelium of 6 patients with limbal stem cell deficiency (LSCD) caused by alkali burn or Stevens Johnson Syndrome were examined by lacrimal endoscope. Cadaveric eyelids were fixed and prepared for cross section and stained with HE and antibodies against PCK, Vim, p63α, SCF and c-Kit. Canaliculus tissue was separated under an operating microscope using a lacrimal probe as an indicator and digested with collagenase A. The clusters of epithelial cells with closely associated stroma were further digested with Trypsin/EDTA to obtain single cells for culture on Matrigel-coated plastic plates in MESCM media. The expression of SCF, c-Kit and p63α was determined by immunostaining. The colony-forming efficiency on 3T3 feeder layers was also measured by calculating the percentage of the clone number divided by the total number cells seeded. Results: The epithelial layers of five out of six inferior lacrimal canaliculi and all the six superior lacrimal canaliculi were visually normal in appearance. Five to fifteen layers of the epithelium in the human lacrimal canaliculi were present with a small, tightly compacted basal layer of cells expressing PCK, p63α, SCF and c-Kit. LCESC were isolated by collagenase A and obtained clonal growth in MESCM. The colony-forming efficiency of LCESC holoclones on a 3T3 feeder layer was 3.2%, compared to 1.9% for those of limbal stem cells (LSC). Conclusions: Herein, we first report that LCESCs can be isolated and have stem cell characteristics, similar to those of LSCs. Such a discovery raises a promising substrate resource of stem cells for LSC reconstruction in LSCD patients.

Gene transfer to human trabecular meshwork cells in vitro and ex vivo using HIV-based lentivirus.

AIM: To investigate whether the enhanced green fluorescent protein (EGFP) reporter gene could be transferred into human trabecular meshwork (HTM) cells by a HIV-based lentivirus both in vitro and ex vivo. METHODS: The HIV-based lentivirus that contains an EF1-α promoter driving EGFP expression cassette was constructed following the standard molecular cloning methods. The cultured HTM cells were transduced at a range of multiplicity of infection (MOI) with HIV-based lentivirus. EGFP positive cell populations were detected by flow cytometry. Human anterior eye segments were cultured with perfusion culture system and transfected by HIV-based lentivirus with a 1×10(8) transducing unit (TU) virus in perfusion liquid. The intraocular pressure was recorded every 8h for 21d. The expression of EGFP in the anterior segment of the human eye was detected by fluorescence microscopy. Furthermore, the distribution of EGFP expression was confirmed by anti-EGFP immunohistochemical staining. RESULTS: The HIV-based lentivirus which contains an EF1-α promoter driving EGFP expression cassette was constructed successfully. After HTM cells were transduced with HIV-based lentivirus containing EGFP in vitro, the ratio of EGFP positive cells to the total cell number reached 92.3%, with the MOI of 15. After the lentivirus containing EGFP were used to transduce human anterior eye segments, the EGFP could be directly detected by fluorescence microscopy in vivo. Immunohistochemistry staining revealed that 88.19% EGFP-positive trabecular meshwork (TM) cells were observed in the human anterior segment. Nevertheless, the intraocular pressure in the lentivirus-transduced group kept constant when compared with control group (P>0.05). CONCLUSION: EGFP gene could be efficiently transferred into HTM cells both in vitro and ex vivo by using HIV-based lentivirus.

Amniotic membrane covering promotes healing of cornea epithelium and improves visual acuity after debridement for fungal keratitis.

AIM: To investigate the effect of amniotic membrane covering (AMC) on the healing of cornea epithelium and visual acuity for fungal keratitis after debridement. METHODS: Twenty fungal keratitis patients were divided into two groups randomly, the AMC group and the control group, ten patients each group. Both debridement of the infected cornea tissue and standard anti-fungus drugs treatments were given to every patients, monolayer amniotic membrane were sutured to the surface of the entire cornea and bulbar conjunctiva with 10-0 nylon suture for patients in the AMC group. The diameter of the ulcer was determined with slit lamp microscope and the depth of the infiltration was determined with anterior segment optical coherence tomography. Uncorrected visual acuity (UCVA) was tested before surgery and three month after healing of the epithelial layer. The healing time of the cornea epithelium, visual acuity (VA) was compared between the two groups using t-test. RESULTS: There was no statistical difference of the diameter of the ulcer, depth of the infiltration, height of the hypopyon and VA between the two groups before surgery (P>0.05). The average healing time of the AMC group was 6.89±2.98d, which was statistically shorter than that of the control group (10.23±2.78d) (P<0.05). The average UCVA of the AMC group was 0.138±0.083, which was statistically better than that of the control group (0.053±0.068) (P<0.05). CONCLUSION: AMC surgery could promote healing of cornea epithelium after debridement for fungal keratitis and lead to better VA outcome.

Promotion of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on titanium.

AIM: To investigate the influence of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on titanium (Ti) surface. METHODS: The chimeric peptide RKLPDAPRGDN (minTBP-1-PRGDN) was synthesized by connecting RKLPDA (minTBP-1) to the N-terminal of PRGDN, the influence of minTBP-1-PRGDN on the attachment, proliferation and collagen I synthesis of human keratocyte on Ti surface were tested using PRGDN and minTBP-1as controls. The keratocytes attached to the surface of Ti were either stained with FITC-labeled phalloidin and viewed with fluorescence microscope or quantified with alamar Blue method. The proliferation of keratocytes on Ti were quantified with 3-(4,5-dim- ethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide up-taking methods. The secretion of type I collagen were determined using an ELISA kit. RESULTS: The results showed that minTBP-1-PRGDN at a concentration of 100ng/mL was the most potent peptide to enhance the attachment of human keratocytes to the surface of Ti (1.40±0.03 folds, P=0.003), to promote the proliferation (1.26±0.05 folds, P=0.014) and the synthesis of type I collagen (1.530±0.128, P=0.008). MinTBP-1 at the same concentration could only promote the attachment (1.13±0.04 folds, P=0.020) and proliferation(1.15±0.06 folds, P=0.021), while PRGDN had no significant influence (P>0.05). CONCLUSION: Our data shows that the novel chimeric peptide minTBP-1-PRGDN could promote the attachment, proliferation and type I collagen synthesis of human keratocytes on the surface of Ti.

[Analysis of anatomy features for patients with narrow anterior chamber angle and primary iridociliary body cysts].

OBJECTIVE: To investigate the prevalence and anatomy features of iridociliary body cysts in patients with narrow anterior chamber angle. METHODS: Retrospective case series study. The prevalence and anatomy features of iridociliary body cysts in 223 patients (402 eyes) were analyzed retrospectively with ultrasound biomicroscopy (UBM). All of the patients were examined for susceptive narrow anterior chamber angle without complaint. The age of the patients, the site, diameter and number of cysts, the anterior chamber angle and the central anterior chamber depth were measured. RESULTS: Iridociliary body cysts were found in 19 patients (23 eyes) out of 223 patients (402 eyes), the prevalence is 5.7%. Fifteen patients were unilateral and four patients bilateral. Two cases originated from the ciliary process, eighteen cases from the iris root, and three from both the root and posterior surface of the iris. Twenty one cases were single cysts while two cases were multiple cysts. The diameter of the cysts ranged from 0.5 to 3.1 mm, averaged (0.71 ± 0.53) mm. The average age and the central anterior chamber depth of the eyes with iridociliary body cysts were (55.32 ± 10.74) years and (2.25 ± 0.39) mm, with no significant difference (t = 0.534, 0.783; P > 0.05) as compared to that of patients without cysts, which were (57.46 ± 10.52) years and (2.14 ± 0.34) mm. The anterior chamber angle in iridociliary body cysts group was 8.2° (21.0°, 0.0°), with no significant difference (Z = -0.062, P > 0.05) as compared to that of patients without cysts, which was 8.9° (21.4°, 0.0°). CONCLUSIONS: The prevalence rate of iridociliary body cysts in this study is 5.7%, central anterior chamber depth and anterior chamber angle in patients with cysts do not differ form patients without cysts.

Mesenchymal stem cells derived from human limbal niche cells.

PURPOSE: We investigated whether human limbal niche cells generate mesenchymal stem cells. METHODS: Limbal niche cells were isolated from the limbal stroma by collagenase alone or following dispase removal of the limbal epithelium (D/C), and cultured on plastic in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), or coated or three-dimensional Matrigel in embryonic stem cell medium with leukemia inhibitory factor and basic fibroblast growth factor. Expression of cell markers, colony-forming units-fibroblast, tri-lineage differentiation, and ability of supporting limbal epithelial stem/progenitor cells were compared to limbal residual stromal cells. RESULTS: Stromal cells expressing angiogenesis markers were found perivascularly, subjacent to limbal basal epithelial cells, and in D/C and limbal residual stromal cells. When seeded in three-dimensional Matrigel, D/C but not limbal residual stromal cells yielded spheres of angiogenesis progenitors that stabilized vascular networks. Similar to collagenase-isolated cells, D/C cells could be expanded on coated Matrigel for more than 12 passages, yielding spindle cells expressing angiogenesis and mesenchymal stem cells markers, and possessing significantly higher colony-forming units-fibroblast and more efficient tri-lineage differentiation than D/C and limbal residual stromal cells expanded on plastic in DMEM with 10% FBS, of which both lost the pericyte phenotype while limbal residual stromal cells turned into myofibroblasts. Upon reunion with limbal epithelial stem/progenitor cells to form spheres, D/C cells expanded on coated Matrigel maintained higher expression of p63α and lower expression of cytokeratin 12 than those expanded on plastic in DMEM with 10% FBS, while spheres formed with human corneal fibroblasts expressed cytokeratin 12 without p63α. CONCLUSIONS: In the limbal stroma, cells subjacent to limbal basal epithelial cells serve as niche cells, and generate progenitors with angiogenesis and mesenchymal stem cells potentials. They might partake in angiogenesis and regeneration during corneal wound healing.

Angiogenesis potential of human limbal stromal niche cells.

PURPOSE: The perivascular localization of stem cell (SC) niches suggests the presence of a vascular niche. We aimed to determine the angiogenesis potential of limbal niche cells (NCs). METHODS: Human limbal NCs were isolated and serially passaged on plastic or coated Matrigel in embryonic SC medium containing BFGF and leukemia inhibitory factor before being reseeded in 3D Matrigel. Expression of angiogenesis markers was assessed by RT-qPCR and immunofluorescence staining. Their angiogenesis potential was measured by differentiation into vascular endothelial cells and by supporting vascular tube network formed by human umbilical vein endothelial cells (HUVEC) on Matrigel. Their support of limbal epithelial progenitor cells (LEPC) was examined in sphere growth formed by reunion in 3D Matrigel. RESULTS: On plastic, limbal NC could be cultured only up to four passages before turning into myofibroblasts. In contrast, on coated Matrigel, they could be expanded for up to 12 passages with upregulation of markers suggestive of angiogenesis progenitors when reseeded in 3D Matrigel because they could differentiate into vascular endothelial cells and pericytes stabilizing the tube network formed by HUVEC. Although both expanded limbal NCs and HUVEC rejoined with LEPC to form spheres to upregulate expression of ΔNp63α, CK15, and CEBPδ, the former but not the latter abolished expression of CK12 keratin. CONCLUSIONS: Human limbal NCs continuously expanded on the basement membrane differentiate into angiogenesis progenitors that prevent differentiation of LEPC/SCs. They may partake in formation of the vascular niche and contribute to angiogenesis during wound healing.

Isolation and expansion of human limbal stromal niche cells.

PURPOSE: Limbal stromal niche cells heterogeneously express embryonic stem cell (SC) markers. This study was conducted to isolate and expand them and to prove that their phenotype is critical for supporting SCs. METHODS: Human limbus was isolated by dispase or collagenase. Single cells were seeded on coated, 2D, or 3D Matrigel and were serially passaged in modified embryonic SC medium (MESCM), supplemented hormonal epithelial medium (SHEM), or Dulbecco's modified Eagle's medium plus 10% fetal bovine serum (DF) before they were seeded in 3D Matrigel. Sphere growth was achieved by mixing expanded single cells with dispase-isolated epithelial cells in 3D Matrigel. Expression of SC markers was analyzed by qRT-PCR, immunofluorescence staining, and Western blot; SC clonal growth was measured on 3T3 feeder layers. RESULTS: Collagenase, but not dispase, isolated subjacent mesenchymal cells, of which the expression of Oct4, Sox2, Nanog, Rex1, SSEA4, N-cadherin, and CD34 was promoted in MESCM more than SHEM or DF. Reunion of PCK+ and Vim+ cells generated spheres in 3D Matrigel, but spindle cells emerged on 2D or coated Matrigel. Serial passages on coated Matrigel resulted in rapid expansion of spindle cells, of which the expression of ESC markers had declined but could be regained after reseeding in 3D Matrigel in MESCM but not in SHEM or DF. Resultant epithelial spheres mixed with spindle cells expanded in MESCM expressed more p63α, less CK12, and more holoclones than those mixed with spindle cells expanded in DF. CONCLUSIONS: Limbal stromal niche cells expressing SC markers can be isolated and expanded to prevent differentiation and maintain clonal growth of limbal epithelial progenitors.

Expression of endoplasmic reticulum stress-related factors in the retinas of diabetic rats.

Recent reports show that ER stress plays an important role in diabetic retinopathy (DR), but ER stress is a complicated process involving a network of signaling pathways and hundreds of factors, What factors involved in DR are not yet understood. We selected 89 ER stress factors from more than 200, A rat diabetes model was established by intraperitoneal injection of streptozotocin (STZ). The expression of 89 ER stress-related factors was found in the retinas of diabetic rats, at both 1- and 3-months after development of diabetes, by quantitative real-time polymerase chain reaction arrays. There were significant changes in expression levels of 13 and 12 ER stress-related factors in the diabetic rat retinas in the first and third month after the development of diabetes, Based on the array results, homocysteine- inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1(HERP), and synoviolin(HRD1) were studied further by immunofluorescence and Western blot. Immunofluorescence and Western blot analyses showed that the expression of HERP was reduced in the retinas of diabetic rats in first and third month. The expression of Hrd1 did not change significantly in the retinas of diabetic rats in the first month but was reduced in the third month.

Limbal epithelial stem/progenitor cells attract stromal niche cells by SDF-1/CXCR4 signaling to prevent differentiation.

Corneal epithelial stem cells (SCs) are an ideal model for investigating how adult lineage-committed epithelial SCs are regulated by an anatomically defined and accessible niche, that is, limbal palisades of Vogt, located between the cornea and the conjunctiva. We have used collagenase digestion to isolate the entire limbal epithelial SCs and subjacent mesenchymal cells, and we have demonstrated that their close association is crucial for promoting epithelial clonal growth, implying that the latter serves as niche cells (NCs). After their close association was disrupted by trypsin/EDTA, single SCs and NCs could reunite to generate sphere growth in three-dimensional Matrigel in the embryonic SC medium, and that such sphere growth initiated by SC-NC reunion was mediated by SDF-1 uniquely expressed by limbal epithelial progenitor cells and its receptor CXCR4, but not CXCR7, strongly expressed by limbal stromal NCs. Inhibition of CXCR4 by AMD3100 or a blocking antibody to CXCR4 but not CXCR7 disrupted their reunion and yielded separate spheres with a reduced size, while resultant epithelial spheres exhibited more corneal differentiation and a notable loss of holoclones. For the first time, these results provide strong evidence supporting that limbal SC function depends on close physical association with their native NCs via SDF-1/CXCR4 signaling. This novel in vitro model of sphere growth with NCs can be used for investigating how limbal SC self-renewal and fate decision might be regulated in the limbal niche.

Effect of DLL4 siRNA on proliferation, migration and tube formation of choroid-retinal endothelial cells under hypoxic conditions.

BACKGROUND: Delta-like 4 (DLL4) is an endothelium specific Notch ligand and has been shown to function as a regulating factor during physiological and pathological angiogenesis. It has been reported that the DLL4-Notch signaling pathway is regulated by hypoxia and may prevent excessive angiogenesis through the inhibition of angiogenic branching and by triggering vessel maturation. Choroidal neovascularization (CNV) is a pathological form of angiogenesis in which hypoxia is thought to play an important role. This study was aimed to evaluate the role of DLL4 in the development of CNV. METHODS: We utilized chemical hypoxia induced by 200 µmol/L CoCl2 to observe the expression of DLL4 in choroid-retinal endothelial cells (RF/6A cells), which are the primary cells involved in CNV. After transfection of a DLL4 small interfering RNA (siRNA), mRNA and protein expression of DLL4 and key downstream genes, including HES1 and HEY1, in hypoxic RF/6A cells were investigated by RT-PCR, real-time PCR, and Western blotting analysis. Three controls were used: one without transfection, one with transfection reagent, and one with scrambled negative control siRNA. The effects of the DLL4 siRNA on the biological function of hypoxic RF/6A cells during angiogenesis, including cell proliferation, migration and tube formation, were investigated. RESULTS: The results showed that hypoxic conditions led to upregulation of DLL4 expression in RF/6A cells in vitro. After transfection, siRNA-duplex1 targeting DLL4 depleted the DLL4 mRNA levels by as much as 91.4% compared with the scrambled siRNA control, and DLL4 protein expression was similarly effected. There was no significant difference in DLL4 expression among the blank control, transfection reagent control, and scrambled siRNA groups. In addition, after transfection of hypoxic RF/6A cells with the DLL4 siRNA-duplex1, the mRNA levels of HES1 and HEY1, which function downstream of DLL4-Notch signaling, were lowered by 75.1% and 86.3%, respectively, compared with the scrambled siRNA control. Furthermore, knockdown of DLL4 expression significantly promoted the proliferation of hypoxic RF/6A cells and led to their arrest in the S phase of the cell cycle. Migration and tube formation of hypoxic RF/6A cells were significantly induced by the DLL4 siRNA, with the number of migrated cells increased by 1.6-fold and total tube length increased by 82.3%, compared with the scrambled siRNA (P < 0.05). CONCLUSIONS: DLL4 functions as a negative regulator of angiogenic branching and sprouting. Based on our results, DLL4 signaling appears to play an essential role in the biological behavior of choroid vascular endothelial cells under hypoxia. Therefore, DLL4 may represent a novel target for CNV therapy in the future.

Redundant mechanisms for vascular growth factors in retinopathy of prematurity in vitro.

BACKGROUND: Current treatments for retinopathy of prematurity (ROP) targeting single vascular growth factors are ineffective in preventing neoangiogenesis. METHODS: We investigated the redundant/compensatory mechanisms between vascular growth factors in ROP. Cultured retinal vascular endothelial cells under CoCl2-induced hypoxia were transfected with recombinant adeno-associated virus type 2-vascular endothelial growth factor (VEGF) or pGIPZ-VEGF RNA interference to up- and downregulate VEGF expression, respectively. At 48 h after transfection, basic fibroblast growth factor (bFGF) and angiopoietin 1 (ANG1) gene expression as well as mitotic cycle changes were analyzed in the cells and correlated with the change in VEGF expression. RESULTS: Compared with the normal control group 1, at 30 min, 12 h and 24 h, the expressions of VEGF, bFGF and ANG1 in the hypoxia control group 2 were significantly higher. In the highly expressing VEGF group (group 3), the expressions of bFGF and ANG1 were downregulated, while in the low-expressing VEGF group 4, the expressions of bFGF and ANG1 were significantly upregulated. In the bevacizumab treatment group 5, the expressions of VEGF, bFGF and ANG1 were similar to those in group 2, and the difference was not significant. CONCLUSIONS: A compensatory mechanism (redundancy) exists between vascular growth factors in ROP. Such a phenomenon could partially explain why the inhibition of a single growth factor cannot effectively prevent the recurrence of neovascularization in ROP. A more effective strategy for treating ROP may be to inhibit VEGF and its redundant pathways.

The role of endoplasmic reticulum stress in the early stage of diabetic retinopathy.

The aim of this study was to evaluate the role of endoplasmic reticulum (ER) stress in diabetic retinopathy (DR) using in vitro and in vivo models. For the in vivo studies, diabetes was induced in rats, and retinal expression of glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), and vascular endothelial growth factor (VEGF) in groups of rats at 1, 3, and 6 months was assessed. For the in vitro studies, human retinal capillary endothelial cells (HRCECs) were cultured in the presence of varying glucose concentrations, and the expression of mRNA and protein for GRP78, ATF4, CHOP, and VEGF was assessed. The chosen glucose concentrations were reflective of those apparent in diabetic patients. Expression of VEGF and CHOP mRNA levels were significantly increased in diabetic rats compared to control rats at 1, 3, and 6 months (P < 0.05). In HRCECs cultured in the presence of high as well as variable glucose concentrations, CHOP expression and apoptosis were significantly increased (P < 0.05). However, GRP78 and ATF4 expression levels were unchanged. Our findings suggest that early progression of DR may be mediated by ER stress, but probably does not involve changes in ATF4 or GRP78.

Effects of endothelin-1 on the cytoskeleton protein F-actin of human trabecular meshwork cells in vitro.

AIM: To observe the effect of endothelin-1 (ET-1) on the cytoskeleton protein F-actin of cultured human trabecular meshwork (HTM) cells. METHODS: CULTURED HTM CELLS WERE RANDOMLY DIVIDED INTO FOUR GROUPS: control group, low-dose ET-1 (10(-9) mol/L) treatment group, middle-dose ET-1 (10(-8) mol/L) treatment group, and high-dose ET-1(10(-7) mol/L) treatment group. After treated with ET-1, the expression of cytoskeleton protein F-actin in trabecular meshwork was analyzed with Western-blot and the distribution of F-actin was detected with FITC-Phalloidin probe. RESULTS: ET-1 dose-dependently and significantly increased F-actin in trabecular meshwork cells (P<0.05). The F-actin stress fiber and periphery actin fiber highly increased and manifested mild reorganization after treated with ET-1; and there were much more cell-to-cell and cell-to-extracellular matrix attachments formation in ET-1 treated HTM cells than that in the untreated HTM cells. CONCLUSION: ET-1 promotes the expression of cytoske- leton protein F-actin and induced the trabecular meshwork actin cytoskeleton reorganization.

The influence of He-Ne laser on scar formation after trabeculectomy in rabbits.

AIM: To investigate the influence of He-Ne lasers on scar formation in the filtration canal after trabeculectomy in a rabbit model, as well as to explore the mechanisms for preventing scar formation when using He-Ne lasers in vivo. METHODS: Experiment 1: Four groups were established (four eyes in each group). In 12 eyes, the upper nasal limbus area next to the upper rectus muscle received 10 minutes of He-Ne laser irradiation (100, 150, 200mW/cm(2); 60, 90, 120J/cm(2)) every day for three days. Four eyes served as controls. Twenty-four hours after the final irradiation, the rabbits were sacrificed and the irradiated tissue was excised, fixed with paraformaldehyde and tested for proliferating cell nuclear antigen (PCNA), connective tissue growth factor (CTGF) and apoptosis (TUNEL). Experiment 2: Forty-two rabbits were randomly divided into two groups and standard trabeculectomy was performed in the right eyes either after 200mW/cm(2) He-Ne laser irradiation or not in the filtration area. The expression of PCNA and CTGF, apoptosis and collagen density in the filtration area were tested on the 7(th), 14(th) and 28(th) day after surgery. RESULTS: Experiment 1: There were no more PCNA and CTGF positive cells in the He-Ne irradiation group than in the control group. No apoptotic cells were found in either group. Experiment 2: The expression of PCNA and CTGF was lower in the He-Ne irradiation group than in the control group on the 7(th) and 14(th) day after trabeculectomy surgery (P<0.05); no apoptotic cells were detected in either group. Collagen density was significantly lower in the He-Ne irradiation group than in the control group on the 14(th) and 28(th) day after surgery (P<0.05). CONCLUSION: Pretreating the filtration area with 200mW/cm(2) (120J/cm(2)) of He-Ne laser irradiation may be helpful in preventing scar formation after trabeculectomy, possibly due to the downregulation of the expression of PCNA, CTGF and collagen synthesis in fibroblasts.

Influence and mechanism of He-Ne laser on scar formation of filtration canal after trabeculectomy in rabbit.

AIM: To investigate the influence of He-Ne laser on connective tissue growth factor (CTGF) expression and collagen formation of fibroblast in filtration site after trabeculectomy in rabbit, and to discuss the mechanism for preventing scar formation with He-Ne laser in vivo. METHODS: The upper nasal limbus area next to the upper rectus muscle in right eyes received 10 minutes He-Ne laser irradiation (200mW/cm(2)) every day for three days, the left eyes served as control. Twenty-four hours after the last irradiation, both eyes of the rabbits were took trabeculectomy surgery. The expressions of CTGF in the filtration area were tested on the 7(th), 14(th) and 28(th) day after surgery and collagen density was tested on the 14(th) and 28(th) day after surgery. Each of the time point had 7 rabbits. RESULTS: The expression of CTGF was lower than that of the control group's on the 7(th) and 14(th) day after trabeculectomy surgery (P=0.01, P=0.005). When examined on the 14(th) and 28(th) day, the collagen density of irradiation group were significantly lower than that of the control group's (P=0.013, P=0.01). CONCLUSION: Pretreating the filtration area with 200mW/cm(2) He-Ne laser may be helpful in preventing scar formation after trabeculectomy in rabbit, possibly due to downregulation of the expression of CTGF and collagen synthesis in fibroblasts. He-Ne laser may be developed into a new scar preventing method in filtration surgery.

[Analysis of multifocal electroretinogram first-order kernel P(1) wave in anisometropic amblyopia].

OBJECTIVE: To investigate the features of the multifocal electroretinogram (mfERG) first-order kernel P(1) wave in anisometropic amblyopic eyes. METHODS: mfERG of fifteen anisometropic amblyopic eyes and their normal control eyes were recorded with ROLAND RETIscan visual evoked response system, the mean amplitude density and eclipse period of the P(1) wave were analyzed and compared between the amblyopic and normal eyes with t-test. RESULTS: The P(1) wave amplitude density of mfERG first-order kernel in amblyopic eyes (164.7 ± 73.1) nV×deg(-2) was significantly attenuated in the central region of the visual field as compared with that of the control eyes (227.0 ± 61.3) nV×deg(-2) (t = 2.554, P = 0.016). The eclipse period of the amblyopic eyes (30.3 ± 4.3) ms was shorter than that of the control eyes (34.4 ± 3.2) ms (t = 2.92, P = 0.007). CONCLUSIONS: The significant change of the multifocal electroretinogram (mfERG) first-order kernel P(1) wave may reflect the abnormality of the retinal on-bipolar cells function and the visual information transmission, whether the P(1) wave could be used as an objective index of amblyopic eyes is a promising research topic.