Qiuli Fu

Generation of Functional Lentoid Bodies From Human Induced Pluripotent Stem Cells Derived From Urinary Cells

PurposenThe pathological mechanisms underlying cataract formation remain largely unknown on account of the lack of appropriate in vitro cellular models. The aim of this study is to develop a stable in vitro system for human lens regeneration using pluripotent stem cells.nnnMethodsnIsolated human urinary cells were infected with four Yamanaka factors to generate urinary human induced pluripotent stem cells (UiPSCs), which were induced to differentiate into lens progenitor cells and lentoid bodies (LBs). The expression of lens-specific markers was examined by real-time PCR, immunostaining, and Western blotting. The structure and magnifying ability of LBs were investigated using transmission electron microscopy and observing the magnification of the letter X, respectively.nnnResultsnWe developed a fried egg differentiation method to generate functional LBs from UiPSCs. The UiPSC-derived LBs exhibited crystalline lens-like morphology and a transparent structure and expressed lens-specific markers αA-, αB-, β-, and γ-crystallin and MIP. During LB differentiation, the placodal markers SIX1, EYA1, DLX3, PAX6, and the specific early lens markers SOX1, PROX1, FOXE3, αA-, and αB-crystallin were observed at certain time points. Microscopic examination revealed the presence of lens epithelial cells adjacent to the lens capsule as well as both immature and mature fiber-like cells. Optical analysis further demonstrated the magnifying ability (1.7×) of the LBs generated from UiPSCs.nnnConclusionsnOur study provides the first evidence toward generating functional LBs from UiPSCs, thereby establishing an in vitro system that can be used to study human lens development and cataractogenesis and perhaps even be useful for drug screening.

Airborne particulate matter (PM2.5) triggers autophagy in human corneal epithelial cell line

PURPOSEnTo investigate particulate matter (PM2.5)-induced damage to human corneal epithelial cells (HCECs) and to determine the underlying mechanisms.nnnMETHODSnHCECs were exposed to PM2.5xa0at a series of concentrations for various periods. Cell viability was measured by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell proliferation was evaluated via 5-ethynyl-2-deoxyuridine (EdU) analysis, while autophagy was determined by immunofluorescence and Western blot.nnnRESULTSnPM2.5-induced cell damage of HCECs occurred in a time- and dose-dependent manner. Decreased cell viability and proliferation as well as increased apoptosis were observed in HCECs after PM2.5 exposure for 24xa0h. Autophagy in HCECs was slightly inhibited in the early stage (before 4xa0h) of exposure but significantly activated in the late stage (after 24xa0h), as evidenced by a decrease in the former and increase in the latter of the expression of the autophagy-associated markers LC3B, ATG5, and BECN1. Interestingly, rapamycin, an autophagy activator, attenuated early-stage but aggravated late-stage PM2.5-induced cell damage, suggesting that the role of autophagy in HCECs may change over time during PM2.5 exposure. In addition, in the early stage, the expression of LC3B and ATG5 increased in cells co-treated with rapamycin and PM2.5 compared to rapamycin-only or PM2.5-only treated cells, suggesting that autophagy may benefit cell viability after PM2.5 exposure.nnnCONCLUSIONSnThe results indicate the potential role of autophagy in the treatment of PM2.5-induced ocular corneal diseases and provide direct evidence for the cytotoxicity, possibly involving an autophagic process, of PM2.5 in HCECs.

Construction of a Corneal Stromal Equivalent with SMILE-Derived Lenticules and Fibrin Glue

The scarcity of corneal tissue to treat deep corneal defects and corneal perforations remains a challenge. Currently, small incision lenticule extraction (SMILE)-derived lenticules appear to be a promising alternative for the treatment of these conditions. However, the thickness and toughness of a single piece of lenticule are limited. To overcome these limitations, we constructed a corneal stromal equivalent with SMILE-derived lenticules and fibrin glue. In vitro cell culture revealed that the corneal stromal equivalent could provide a suitable scaffold for the survival and proliferation of corneal epithelial cells, which formed a continuous pluristratified epithelium with the expression of characteristic markers. Finally, anterior lamellar keratoplasty in rabbits demonstrated that the corneal stromal equivalent with decellularized lenticules and fibrin glue could repair the anterior region of the stroma, leading to re-epithelialization and recovery of both transparency and ultrastructural organization. Corneal neovascularization, graft degradation, and corneal rejection were not observed within 3u2009months. Taken together, the corneal stromal equivalent with SMILE-derived lenticules and fibrin glue appears to be a safe and effective alternative for the repair of damage to the anterior cornea, which may provide new avenues in the treatment of deep corneal defects or corneal perforations.

Air pollution and outpatient visits for conjunctivitis: A case-crossover study in Hangzhou, China

BACKGROUNDnConjunctivitis, one of the most common ocular surface diseases, can be caused by many factors.nnnOBJECTIVESnThe objective of this study was to investigate the potential association between conjunctivitis and air pollutants.nnnMATERIALS AND METHODSnData of 9737 outpatient visits for conjunctivitis from July 1, 2014 to June 30, 2016 were obtained from the Eye Center of the Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China. The data were linked to data on the concentrations of carbon monoxide (CO), nitrogen dioxide (NO2), ozone (O3), sulfur dioxide (SO2), and fine particulate matter with a median aerometric diameter of less than 10 and 2.5xa0μm (PM10 and PM2.5, respectively), which were obtained from the Environmental Protection Department of Zhejiang Province. A time-stratified case-crossover study design and conditional logistic regression were applied to analyze the association between air pollutants and outpatient visits for conjunctivitis.nnnRESULTSnA 10xa0μg/m3 increase in PM10, PM2.5, SO2, NO2, and CO concentrations on the same day as the hospital visit or on lag days before the hospital visit date was associated with outpatient visits for conjunctivitis. The strongest association was observed between SO2 and conjunctivitis patients aged 2-5 years. Variation occurs between warm and cold seasons, between genders, and among different age groups.nnnCONCLUSIONSnOur study provided evidence that outpatient visits for conjunctivitis were significantly associated with air pollution in Hangzhou, China.

Transcorneal electrical stimulation promotes survival of retinal ganglion cells after optic nerve transection in rats accompanied by reduced microglial activation and TNF-α expression.

Microglial activation plays a crucial role in the pathological processes of various retinal and optic nerve diseases. TNF-α is a pro-inflammatory cytokine that is rapidly upregulated and promotes retinal ganglion cells (RGCs) death after optic nerve injury. However, the cellular source of TNF-α after optic nerve injury remains unclear. Thus, we aimed to determine the changes of retinal microglial activation in a rat model of optic nerve transection (ONT) after transcorneal electrical stimulation (TES). Furthermore, we assessed TNF-α expression after ONT and evaluated the effects of TES on TNF-α production. Rats were divided into 2 control groups receiving a sham surgery procedure, 2 ONT+Sham TES groups, and 2 ONT+TES groups. The rats were sacrificed on day 7 or 14 after ONT. RGCs were retrogradely labelled by Fluorogold (FG) 7 days before ONT, one TES group and corresponding controls were stimulated on day 0, 4, and the second were stimulated on day 0, 4, 7, 10. Whole-mount immunohistofluorescence, quantification of RGCs and microglia, and western blot analysis were performed on day 7 and 14 after ONT. TES significantly increased RGCs survival on day 7 and 14 after ONT, which was accompanied by reduced microglia on day 7, but not 14. TNF-α was co-localized with ameboid microglia and significantly increased on day 7 and 14 after ONT. TES significantly reduced TNF-α production on day 7 and 14 after ONT. Our study demonstrated that TES promotes RGCs survival after ONT accompanied by reduced microglial activation and microglia-derived TNF-α production.

Acute effects of air pollution on respiratory disease mortalities and outpatients in Southeastern China

The objective of this study was to investigate the potential association between air pollutants and respiratory diseases (RDs). Generalized additive models were used to analyze the effect of air pollutants on mortalities or outpatient visits. The average concentrations of air pollutants in Hangzhou (HZ) were 1.6–2.8 times higher than those in Zhoushan (ZS), except for O3. In a single pollutant model, the increased concentrations of PM2.5, NO2, and SO2 were strongly associated with deaths caused by RD in HZ, while PM2.5 and O3 were associated with deaths caused by RD in ZS. All air pollutants (PM2.5, NO2, SO2, and O3) were strongly associated with outpatient visits for RD in both HZ and ZS. In multiple pollutant models, a significant association was only observed between PM2.5 and the mortality rate of RD patients in both HZ and in ZS. Moreover, strong associations between SO2, NO2, and outpatient visits for RD were observed in HZ and ZS. This study has provided evidence that both the mortality rates and outpatient visits for RD were significantly associated with air pollutants. Furthermore, the results showed that different air pollutant levels lead to regional differences between mortality rates and outpatient visits.

A New Long Noncoding RNA ALB Regulates Autophagy by Enhancing the Transformation of LC3BI to LC3BII during Human Lens Development

Autophagy is essential in lens organelle degradation. This study aimed to seek potential autophagy-associated long noncoding RNAs (lncRNAs) and their relative mechanisms in human lens development using the “fried egg” lentoid body (LB) generation system. The expression pattern of LC3B in differentiating LBs was similar to that in developing a mouse lens in vivo. Among the massive lncRNAs expressed with a significant difference between induced pluripotent stem cells (iPSCs) and LBs, lncRNA affecting LC3B (ALB), which was predicted to have a co-relationship with the autophagy marker LC3B, was highly expressed in differentiating lens fibers in LBs. This result was consistent with its high expression in human embryonic lenses compared to those in embryonic stem cells (ESCs). Furthermore, lncRNA ALB knockdown resulted in the downregulation of LC3BII at the protein level, therefore inhibiting the autophagy process in human lens epithelial cells (HLECs). Our results identify lncRNA ALB, a potential autophagy regulator in organelle degradation during human lens development, highlighting the importance of lncRNAs in lens development.

Effects of senescent lens epithelial cells on the severity of age-related cortical cataract in humans: A case-control study.

AbstractThe aging of lens progenitor cell has been repeatedly proposed to play a key role in age-related cataracts (ARCs), but the mechanism is far from being understood. The present study aims to investigate the relationship between aging of lens progenitor/epithelial cells and the 4 subtypes of ARCs in humans.Lens capsules, which were collected from ARC patients during surgery, were divided into 3 groups according to the age of patients (50–60, 60–80, and >80 years). The expressions of lens progenitor cell-related markers Sox2, Abcg2, and Ki67 were first examined in human lens epithelial cells (HLECs) in situ. Then, the percentage of senescent and SA-&bgr;-gal+ HLECs isolated from lens capsules were quantified. Finally, the potential relationships between the percentage of senescent (and SA-&bgr;-gal+) HLECs and the severity of ARCs were analyzed.Ki67+, Sox2+, and Abcg2+ HLECs in lens capsules were clearly more abundant in young people than in patients older than 50 years, and they were almost absent in patients older than 60 years. The percentage of primary HLECs with aging morphology increased with age, consistent with the results of SA-&bgr;-gal+ primary HLECs. Only cortical cataract classification was found to be strongly related to the percentage of SA-&bgr;-gal+ and senescent HLECs.Our study gave the initial evidence on the dynamical change of lens stem/progenitor cells in human lens capsule with age and suggested that lens progenitor/epithelial cell aging is important in the severity of cortical cataracts.

Proliferative Effects of Histamine on Primary Human Pterygium Fibroblasts

Purpose. It has been confirmed that inflammatory cytokines are involved in the progression of pterygium. Histamine can enhance proliferation and migration of many cells. Therefore, we intend to investigate the proliferative and migratory effects of histamine on primary culture of human pterygium fibroblasts (HPFs). Methods. Pterygium and conjunctiva samples were obtained from surgery, and toluidine blue staining was used to identify mast cells. 3-[4, 5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was performed to evaluate the proliferative rate of HPFs and human conjunctival fibroblasts (HCFs); ki67 expression was also measured by immunofluorescence analysis. Histamine receptor-1 (H1R) antagonist (Diphenhydramine Hydrochloride) and histamine receptor-2 (H2R) antagonist (Nizatidine) were added to figure out which receptor was involved. Wound healing model was used to evaluate the migratory ability of HPFs. Results. The numbers of total mast cells and degranulated mast cells were both higher in pterygium than in conjunctiva. Histamine had a proliferative effect on both HPFs and HCFs, the effective concentration (10u2009μmol/L) on HPFs was lower than on HCFs (100u2009μmol/L), and the effect could be blocked by H1R antagonist. Histamine showed no migratory effect on HPFs. Conclusion. Histamine may play an important role in the proliferation of HPFs and act through H1R.

Embryonic Surface Ectoderm-specific Mitofusin 2 Conditional Knockout Induces Congenital Cataracts in Mice

Inherited mitochondrial mutations can result in mitochondrial dysfunction or stochastic oxidative damage. Cumulative mitochondrial damage is an important factor in age-related disorders, such as cataracts and macular degeneration. Mfn2 mediates the fusion of mitochondria and contribute to the dynamic balance between fusion and fission that determines mitochondria morphology. We report here that conditional loss of Mfn2 function in the head surface ectoderm leads to a range of congenital eye defects, including small, opacified lens and small eyeball in the most severe phenotypes. The Le-Cre transgenic mouse line and Mfn2 flox mouse line were used in this study to generate Mfn2 conditional knockout mice. Our study revealed Mfn2 gene function in lens development and addressed the relationship between the mitochondria and lens transparency. Conditional loss of Mfn2 affected lens epithelium cell proliferation, apoptosis and ultrastructure of mitochondria. We conclude that proper development of the lens and lens transparency depend on normal Mfn2 gene function.