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Sample records for corneal endothelial cells

  1. Normal corneal endothelial cell density in Nigerians

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    Ewete T

    2016-03-01

    Full Text Available Temitope Ewete,1 Efeoghene Uchenna Ani,2 Adegboyega Sunday Alabi1 1MeCure Eye Center, Lagos, 2Department of Ophthalmology, University of Port Harcourt, Port Harcourt, Nigeria Aim: The aim of the study was to describe the corneal endothelial cell density of adults at the MeCure Eye Center and to determine the relationship between age, sex, and corneal endothelial cell density. Methods: This study was a retrospective study looking at those records of individuals who had undergone specular microscopy or corneal endothelial cell count measurement at the MeCure Eye Center. Results: The endothelial cell characteristics of 359 healthy eyes of 201 volunteers were studied. The mean corneal endothelial cell density (MCD was 2,610.26±371.87 cells/mm2 (range, 1,484–3,571 cells/mm2. The MCD decreased from 2,860.70 cells/mm2 in the 20–30-year age group to 2,493.06 cells/mm2 in the >70-year age group, and there was a statistically significant relationship between age and MCD with a P-value of <0.001. There was no statistically significant correlation between sex and corneal endothelial cell density (P=0.45. Conclusion: This study shows that endothelial cell density in Nigerian eyes is less than that reported in the Japanese, American, and Chinese eyes, and is comparable to that seen in Indian and Malaysian eyes. Keywords: corneal, endothelial cell density, Nigerian

  2. Effects of diabetic keratopathy on corneal optical density, central corneal thickness, and corneal endothelial cell counts.

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    Gao, Feng; Lin, Tao; Pan, Yingzhe

    2016-09-01

    Diabetic keratopathy is an ocular complication that occurs with diabetes. In the present study, the effect of diabetic keratopathy on corneal optical density, central corneal thickness, and corneal endothelial cell count was investigated. One hundred and eighty diabetic patients (360 eyes) were enrolled in the study during the period from March, 2012 to March, 2013. The patients were divided into three age groups: 10 years, with 60 patients per group (120 eyes). During the same period, 60 healthy cases (120 eyes) were selected and labeled as the normal control group. The Pentacam was used to measure the corneal optical density, and central corneal thickness. Specular microscopy was used to examine the corneal endothelial cell density. The coefficient of partial correlation was used to control age and correlate the analysis between the corneal optical density, corneal endothelial cell density, and central corneal thickness. The stage of the disease, the medial and intimal corneal optical density and central corneal thickness was analyzed in the diabetes group. The corneal optical density in the diabetes group increased compared with that of the normal control group. The medial and intimal corneal optical density and central corneal thickness were positively correlated with the course of the disease. However, the corneal endothelial cell density was not associated with the course of diabetes. There was a positive association between the medial and intimal corneal optical density and central corneal thickness of the diabetic patients. In conclusion, the results of the present study show that medial and intimal corneal optical density and central corneal thickness were sensitive indicators for early diabetic keratopathy.

  3. Development of new therapeutic modalities for corneal endothelial disease focused on the proliferation of corneal endothelial cells using animal models.

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    Koizumi, Noriko; Okumura, Naoki; Kinoshita, Shigeru

    2012-02-01

    This review describes our recent attempts to develop new therapeutic modalities for corneal endothelial disease using animal models including non-human primate model in which the proliferative ability of corneal endothelial cells is severely limited, as is the case in humans. First, we describe our attempt to develop new surgical treatments using cultivated corneal endothelial cells for advanced corneal endothelial dysfunction. It includes two different approaches; a "corneal endothelial cell sheet transplantation" with cells grown on a type-I collagen carrier, and a "cell-injection therapy" combined with the application of Rho-kinase (ROCK) inhibitor. Recently, it was reported that the selective ROCK inhibitor, Y-27632, promotes cell adhesion and proliferation and inhibits the apoptosis of primate corneal endothelial cells in culture. When cultivated corneal endothelial cells were injected into the anterior chamber of animal eyes in the presence of ROCK inhibitor, endothelial cell adhesion was promoted and the cells achieved a high cell density and a morphology similar to corneal endothelial cells in vivo. We are also trying to develop a novel medical treatment for the early phase of corneal endothelial disease by the use of ROCK inhibitor eye drops. In rabbit and monkey experiments using partial endothelial dysfunction models, corneal endothelial wound healing was accelerated by the topical application of ROCK inhibitor to the ocular surface, and resulted in the regeneration of a corneal endothelial monolayer with a high endothelial cell density. We are now trying to advance the clinical application of these new therapies for patients with corneal endothelial dysfunction.

  4. Mechanism of Corneal Endothelial Cells Lesion during Phacoemulsification and Aspiration

    Institute of Scientific and Technical Information of China (English)

    Songtao Yuan; Lina Xie; Qinghuai Liu; Nanrong Yuan

    2003-01-01

    Purpose: To evaluate the proportions of corneal endothelial lesion caused by differentfactors during phacoemulsification and aspiration.Methods: Fourteen cats (twenty eight eyes) were divided into four groups. The processedfactors were ultrasonic power, lens extraction by phacoemulsification or not, and lensextraction using different levels of ultrasonic power. The density of central cornealendothelial cells was measured before and after operation.Results: There is no statistic difference between pre-operation density and post-operationdensity for releasing ultrasonic power only without lens extraction group. But for the lensextraction group, there is difference in density of central corneal endothelial cells andthe higher level of ultrasonic power, the more the central corneal endothelial cells densitydecreased through operation.Conclusion: The primary factor that causes corneal endothelial lesion duringphacoemulsification and aspiration procedure is debris of lens nucleus, and the otherfactors cause the lesion of corneal endothelium in normal operations just in very smalldegree.

  5. Mechanism of induction of fibroblast to corneal endothelial cell.

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    Jiang, Yan; Fu, Wei-Cai; Zhang, Lin

    2014-08-01

    To explore mechanism of nduction of fibroblast to corneal endothelial cell. Rabbit conjunctiva fibroblasts were used as feeder cells, rabbit oral mucosa epithelial cells were used as seed cells, and human denuded amniotic membrane was used as carrier to establish tissue engineering corneal endothelium. The transformation effect was observed. As concentration of mitomycin C increased, cell survival rate gradually decreased, cell proliferation was obviously inhibited when concentration≥25 μg/mL; 5 days after being treated by 5 μg/mL mitomycin C, cell body was enlarged and extended without cell fusion, however after being treated by 0.5 μg/mL mitomycin C, cell body was significantly proliferated and gradually fused; after 3 weeks of culture, stratified epithelium appeared on rabbit oral mucosa epithelial cells, differentiation layers were 4-5 and were well differentiated, the morphology was similar to corneal endothelial cells; Under electron microscope, surface layer of cells were polygonal, tightly connected to another with microvilli on the border, there was hemidesmosome between basal cells and human denuded amniotic membrane. Fibroblast cells have the potential of multi-directional differentiation, effective induction can promote emergence of intercellular desmosomes between seed cells and emergence of epithelial surface microvilli, and differentiate to the corneal endothelial cell. However, clinical application still needs more research and safety evaluation. Copyright © 2014 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

  6. Cartographic system for spatial distribution analysis of corneal endothelial cells.

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    Corkidi, G; Márquez, J; García-Ruiz, M; Díaz-Cintra, S; Graue, E

    1994-07-01

    A combined cartographic and morphometric endothelium analyser has been developed by integrating the HISTO 2000 histological imaging and analysis system with a prototype human corneal endothelium analyser. The complete system allows the elaboration and analysis of cartographies of corneal endothelial tissue, and hence the in vitro study of the spatial distribution of corneal endothelial cells, according to their regional morphometric characteristics (cell size and polygonality). The global cartographic reconstruction is obtained by sequential integration of the data analysed for each microscopic field. Subsequently, the location of each microscopically analysed field is referred to its real position on the histologic preparation by means of X-Y co-ordinates; both are provided by micrometric optoelectronic sensors installed on the optical microscope stage. Some cartographies of an excised human corneal keratoconus button in vitro are also presented. These cartographic images allow a macroscopic view of endothelial cells analysed microscopically. Parametric colour images show the spatial distribution of endothelial cells, according to their specific morphometric parameters, and exhibit the variability in size and cellular shape which depend on the analysed area.

  7. Experiment Study of Effect of Perfiuorohexyloctane on Corneal Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    Xiaoyan Ding; Chunfang Li; Lin Lu; Guanguang Feng; Huling Zheng

    2001-01-01

    Purpose: To investigate the effect of Perfluorohexyloctane (F6H8)on corneal endothelial celIs(CEC) of rabbit eyes. Methods: Fifteen New Zealand white rabbits were devided into two groups:experimental group(F6H8) and control group(BSS) . All rabbits underwent anterior chamber injection of 0. 15ml F6H8 or BSS. Slit-lamp biomicroscopy and corneal endothelium photography were performed pre-operatively and postoperatively. Histopathological examination and Transmission electron microscopy(TEM) were done after the rabbits were sacrificed. Results: All the corneas were clear. Since 4 weeks after operation, the endothelial cells were markedly irregular in size and shape and the number of endothelial cells was markedly decreased. Multilayered retrocorneal membranes (RCM)grew gradually 2 weeks after surgery. Vacuolar degeneration was seen in some endothelial cells. Nuclear degeneration and edema of plasma were seen in TEM. Conclusion: Corneal endothelial cell degenerated after contacting with F6H8 for 2 ~4weeks. As a silicone solvent, it should be removed completely after injection. We don't recommend it to be used as a new intraocular temponade. Eye Science 2001: 17:21 ~ 26.

  8. Morphological changes in corneal endothelial cells after penetrating keratoplasty.

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    Laing, R A; Sandstrom, M; Berrospi, A R; Leibowitz, H M

    1976-09-01

    Fifteen patients who had had a successful penetrating keratoplasty were photographed with the clinical specular microscope and the resulting endothelial photomicrographs were analyzed. The average endothelial cell area was one to six times larger and the average endothelial cell perimeter was one to 2 1/2 times larger than that of a normal cornea of a subject the same age as the donor. In each corneal graft, endothelial cell areas and perimeters clustered tightly around a mean value, although the mean value for different corneas varied significantly. The thickness and transparency of each graft was normal, indicating that within the observed limits the success of the transplantation procedure did not depend on final endothelial cell size or perimeter.

  9. Corneal Endothelial Cell Density and Morphology in Healthy Turkish Eyes

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    Ceyhun Arıcı

    2014-01-01

    Full Text Available Purpose. To describe the normative values of corneal endothelial cell density, morphology, and central corneal thickness in healthy Turkish eyes. Methods. Specular microscopy was performed in 252 eyes of 126 healthy volunteers (M : F, 42 : 84. Parameters studied included mean endothelial cell density (MCD, mean cell area (MCA, coefficient of variation (CV in cell size, percentage of hexagonal cells, and central corneal thickness (CCT. Results. The mean age of volunteers was 44.3±13.5 (range, 20 to 70 years. There was a statistically significant decrease in MCD (P<0.001; correlation, −0.388 and percentage of hexagonal cells, (P<0.001; correlation, −0.199 with age. There was also a statistically significant increase in MCA (P<0.001; correlation, 0.363 with increasing age. There was no statistically significant difference in MCD, MCA, CV in cell size, percentage of hexagonal cells, and CCT between genders and there was also no significant difference in these parameters between fellow eyes of subjects. Conclusions. Normotive data for the endothelium in the Turkish population are reported. Endothelial cell density in the Turkish eyes is less than that described in the Japanese, American, Chinese, and Filipino eyes and higher than that described in Indian, Thai, and Iranian eyes.

  10. Corneal endothelial cell density and morphology in Phramongkutklao Hospital

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    Narumon Sopapornamorn

    2008-03-01

    Full Text Available Narumon Sopapornamorn1, Manapon Lekskul1, Suthee Panichkul21Department of Ophthalmology, Phramongkutklao Hospital, Bangkok, Thailand; 2Department of Obstetrics and Gynecology, Phramongkutklao College of Medicine, Bangkok, ThailandObjective: To describe the corneal endothelial density and morphology in patients of Phramongkutklao Hospital and the relationship between endothelial cell parameters and other factors.Methods: Four hundred and four eyes of 202 volunteers were included. Noncontact specular microscopy was performed after taking a history and testing the visual acuity, intraocular pressure measurement, Schirmer’s test and routine eye examination by slit lamp microscope. The studied parameters included mean endothelial cell density (MCD, coefficient of variation (CV, and percentage of hexagonality.Results: The mean age of volunteers was 45.73 years; the range being 20 to 80 years old. Their MCD (SD, mean percentage of CV (SD and mean (SD percentage of hexagonality were 2623.49(325 cell/mm2, 39.43(8.23% and 51.50(10.99%, respectively. Statistically, MCD decreased significantly with age (p < 0.01. There was a significant difference in the percentage of CV between genders. There was no statistical significance between parameters and other factors.Conclusion: The normative data of the corneal endothelium of Thai eyes indicated that, statistically, MCD decreased significantly with age. Previous studies have reported no difference in MCD, percentage of CV, and percentage of hexagonality between gender. Nevertheless, significantly different percentages of CV between genders were presented in this study.Keywords: Corneal endothelial cell, parameters, age, gender, smoking, Thailand

  11. Effect of Mitomycin-C augmented trabeculectomy on corneal endothelial cells

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    Reza Zarei

    2015-01-01

    Conclusion: MMC application in trabeculectomy seems to cause a small but significant corneal endothelial loss. Most of the damage occurs intraoperatively, or in the early postoperative period, however progressive endothelial cell loss is not a major concern.

  12. Corneal endothelial cell changes associated with cataract surgery in patients with type 2 diabetes mellitus

    DEFF Research Database (Denmark)

    Hugod, Mikkel; Storr-Paulsen, Allan; Norregaard, Jens Christian;

    2011-01-01

    To investigate the corneal endothelial cell density and morphology in patients with and without diabetes after phacoemulsification with intraocular lens implantation.......To investigate the corneal endothelial cell density and morphology in patients with and without diabetes after phacoemulsification with intraocular lens implantation....

  13. Discovery of molecular markers to discriminate corneal endothelial cells in the human body

    NARCIS (Netherlands)

    Yoshihara, Masahito; Ohmiya, Hiroko; Hara, Susumu; Kawasaki, Satoshi; Hayashizaki, Yoshihide; Itoh, Masayoshi; Kawaji, Hideya; Tsujikawa, Motokazu; Nishida, Kohji; Clevers, J.C.; van de Wetering, M.L.

    2015-01-01

    The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant

  14. Discovery of molecular markers to discriminate corneal endothelial cells in the human body

    NARCIS (Netherlands)

    Yoshihara, Masahito; Ohmiya, Hiroko; Hara, Susumu; Kawasaki, Satoshi; Hayashizaki, Yoshihide; Itoh, Masayoshi; Kawaji, Hideya; Tsujikawa, Motokazu; Nishida, Kohji; Clevers, J.C.; van de Wetering, M.L.

    2015-01-01

    The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant nu

  15. Corneal endothelial cell density and morphology and central corneal thickness in Guangxi Maonan and Han adolescent students of China

    Institute of Scientific and Technical Information of China (English)

    Hao; Liang; Hui-Yi; Zuo; Jin-Mao; Chen; Jie; Cai; Yu-Zhua; Qin; Yu-Ping; Huang; Ying-Ying; Chen; Dong-Yong; Tang; Shao-Jian; Tan

    2015-01-01

    AIM: To investigate the corneal endothelial cell density and morphology and central corneal thickness in the Guangxi Maonan and Han adolescent students of China.METHODS: Noncontact specular microscope(Topcon SP3000 P, Tokyo, Japan) was performed in 133 adolescent students of Maonan nationality(M:F 54:79)and 105 adolescent students of Han nationality(M:F 50:55),5 to 20 y of age, who were randomly selected from 3schools in Huanjiang Maonan Autonomous County of Guangxi Zhuang Autonomous Region of China.Parameters studied included endothelial cell density,mean cell area, coefficient of variation in cell size,percentage hexagonality and central corneal thickness. RESULTS: Endothelial cell density, mean cell area,coefficient of variation in cell size, percentage hexagonality and central corneal thickness in the study population were(2969.50 ±253.93) cells/mm2,(339.23 ±29.44) μm2,(29.96 ±4.07) %,(64.58 ±9.41) % and(523.71 ±32.82) μm in Maonan and(2998.26 ±262.65) cells/mm2,(336.11±30.07) μm2,(29.89±5.03) %,(64.91±11.64) % and(524.39 ±33.15) μm in Han, respectively. No significant differences were observed in endothelial cell density,mean cell area, coefficient of variation in cell size,percentage hexagonality and central corneal thickness between Maonan and Han(P =0.615, 0.659, 0.528, 0.551,0.999). In Maonan and Han, we found age was negatively correlated with endothelial cell density and percentagehexagonality and positively correlated with mean cell area and coefficient of variation in cell size. Negative correlation was also found between central corneal thickness and age in Han, whereas no correlation was found in Maonan. CONCLUSION: There were no differences between Maonan and Han in corneal endothelial cell density and morphology and central corneal thickness. In these two nationalities, there were statistically significant decrease in endothelial cell density and percentage hexagonality with increasing age and statistically significant increase in

  16. Nerve growth factor modulate proliferation of cultured rabbit corneal endothelial cells and epithelial cells.

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    Li, Xinyu; Li, Zhongguo; Qiu, Liangxiu; Zhao, Changsong; Hu, Zhulin

    2005-01-01

    In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF. MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570 nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner. 50 U/mL and 500 U/mL NGF got more increase of proliferation than that of 5 U/mL NGF did. Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.

  17. N-Isopropylacrylamide-co-glycidylmethacrylate as a Thermoresponsive Substrate for Corneal Endothelial Cell Sheet Engineering

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    Bernadette K. Madathil

    2014-01-01

    Full Text Available Endothelial keratoplasty is a recent shift in the surgical treatment of corneal endothelial dystrophies, where the dysfunctional endothelium is replaced whilst retaining the unaffected corneal layers. To overcome the limitation of donor corneal shortage, alternative use of tissue engineered constructs is being researched. Tissue constructs with intact extracellular matrix are generated using stimuli responsive polymers. In this study we evaluated the feasibility of using the thermoresponsive poly(N-isopropylacrylamide-co-glycidylmethacrylate polymer as a culture surface to harvest viable corneal endothelial cell sheets. Incubation below the lower critical solution temperature of the polymer allowed the detachment of the intact endothelial cell sheet. Phase contrast and scanning electron microscopy revealed the intact architecture, cobble stone morphology, and cell-to-cell contact in the retrieved cell sheet. Strong extracellular matrix deposition was also observed. The RT-PCR analysis confirmed functionally active endothelial cells in the cell sheet as evidenced by the positive expression of aquaporin 1, collagen IV, Na+-K+ ATPase, and FLK-1. Na+-K+ ATPase protein expression was also visualized by immunofluorescence staining. These results suggest that the in-house developed thermoresponsive culture dish is a suitable substrate for the generation of intact corneal endothelial cell sheet towards transplantation for endothelial keratoplasty.

  18. Optimization of Human Corneal Endothelial Cells for Culture: The Removal of Corneal Stromal Fibroblast Contamination Using Magnetic Cell Separation

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    Gary S. L. Peh

    2012-01-01

    Full Text Available The culture of human corneal endothelial cells (CECs is critical for the development of suitable graft alternative on biodegradable material, specifically for endothelial keratoplasty, which can potentially alleviate the global shortage of transplant-grade donor corneas available. However, the propagation of slow proliferative CECs in vitro can be hindered by rapid growing stromal corneal fibroblasts (CSFs that may be coisolated in some cases. The purpose of this study was to evaluate a strategy using magnetic cell separation (MACS technique to deplete the contaminating CSFs from CEC cultures using antifibroblast magnetic microbeads. Separated “labeled” and “flow-through” cell fractions were collected separately, cultured, and morphologically assessed. Cells from the “flow-through” fraction displayed compact polygonal morphology and expressed Na+/K+ATPase indicative of corneal endothelial cells, whilst cells from the “labeled” fraction were mostly elongated and fibroblastic. A separation efficacy of 96.88% was observed. Hence, MACS technique can be useful in the depletion of contaminating CSFs from within a culture of CECs.

  19. Long-term corneal endothelial cell changes in pediatric intraocular lens reposition and exchange cases.

    Science.gov (United States)

    Wang, Yan; Wu, Mingxing; Zhu, Liyuan; Liu, Yizhi

    2012-04-01

    To evaluate long-term corneal endothelial cell changes of intraocular lens (IOL) reposition and exchange in children. State key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, 510060, China In this retrospective study, all IOL reposition and exchange procedures performed in patients under 14 years old between January 1999 and April 2009 were included. Follow-up outcomes included corneal endothelial cell density, hexagonality, coefficient of variance, average cell size. IOL reposition procedures in 12 eyes (12 cases) (reposition group, RPG), and IOL exchanges in eight eyes (eight cases) (exchange group, EXG) were performed because of IOL pupillary capture or IOL dislocation. Median of follow-up was 44.5 months in RPG and 66.2 months in EXG. The density of corneal endothelial cells in RPG (2,053 ± 493/mm(2)) and EXG (2,100 ± 758/mm(2)) was significantly decreased in comparison to the control eyes (3,116 ± 335/mm(2)). Hexagonality of corneal endothelial cells and coefficient of variance showed no difference among the control group, RPG and EXG (P > 0.05). The density of corneal endothelial cells was conspicuously decreased after IOL reposition or exchange procedures in childhood cases. Longer follow-up must be conducted in these cases.

  20. Effects of phacoemulsification on the intraocular pressure and corneal endothelial cells of the patients with glaucoma

    Institute of Scientific and Technical Information of China (English)

    Ye Zhao; Zhi-Feng Liu

    2016-01-01

    Objective:To explore the intraocular pressure and corneal endothelial cells integrity changes in cataract phacoemulsification after anti-glaucoma surgery.Methods:Phacoemulsification was performed in 102 patients (118 eyes) with cataract after anti-glaucoma surgery and the intraocular pressure and corneal endothelial cell integrity changes of patients were observed at Day 1 and 3, first week and first month before and after surgery, including central corneal endothelial cell density, average cell area (AVE), cell area of coefficient of variation (CV) and central corneal thickness (CCT).Results:The intraocular pressure was elevated, the central endothelial cell density was reduced, the AVE, the CV and CTT thicken were increased at Day 1 and 3, first week and first month after surgery. The difference compared with preoperative was statistically significant. The intraocular pressure and CTT almost recovered to preoperative levels in 1 month after cataract phacoemulsification and the difference was not statistically significant; while the central endothelial cell density was still decreased and AVE and CV were still increased and the difference of these indexes and the coefficient of the patients was statistically significant compared with before surgery.Conclusions:For the patients with anti-glaucoma after cataract phacoemulsification, intraocular pressure and endothelial cell integrity change was initially observed at Day 1 after surgery, whereas they can almost return to the preoperative level in a month after surgery.

  1. [Morphometric changes of corneal endothelial cells in pseudoexfoliation syndrome and pseudoexfoliation glaucoma].

    Science.gov (United States)

    de Juan-Marcos, L; Cabrillo-Estévez, L; Escudero-Domínguez, F A; Sánchez-Jara, A; Hernández-Galilea, E

    2013-11-01

    To evaluate the corneal endothelial morphometry and central corneal thickness (CCT) in pseudoexfoliative (PEX) eyes with and without glaucoma and to compare with normal eyes and eyes with primary open-angle glaucoma (POAG). A total of 166 patients were included in this study: 36 eyes with pseudoexfoliation syndrome (PXS), 30 eyes with pseudoexfoliation glaucoma (PXG), 40 eyes with POAG, and 60 normal eyes. Corneal endothelial cell density (ECD), coefficient of variation (CV) in cell size, and percentage of hexagonal cells, were measured using a non-contact specular microscope, whereas CCT was measured with an ultrasonic pachymeter. ECD and percentage of hexagonal cells were lower in PEX groups and in the POAG group compared with normal eyes, while the CV in cell size was greater. There was a tendency for greater cell loss and morphological abnormalities of the corneal endothelial cells in PXG eyes compared to PXS eyes, when all pseudoexfoliative eyes were analyzed together. Changes in endothelial cells increased with age. There were no significant differences in mean CCT between the four groups. Endothelial cell density is significantly decreased, and pleomorphism and polymegathism of cells are increased in PEX eyes, particularly when intraocular pressure is high. Copyright © 2012 Sociedad Española de Oftalmología. Published by Elsevier Espana. All rights reserved.

  2. Establishment of a novel corneal endothelial cell line from domestic rabbit, Oryctolagus curiculus.

    Science.gov (United States)

    Fan, TingJun; Zhao, Jun; Fu, YongFeng; Cong, RiShan; Guo, RuiChao; Liu, WanShun; Han, BaoQin; Yu, QiuTao; Wang, Jing

    2007-04-01

    To develop a rabbit corneal endothelial (RCE) cell line, in vitro culture of RCE cells was initiated from Oryctolagus curiculus corneas and a novel RCE cell line was established in this study. To initiate the primary culture of RCE cells, corneas from rabbit eyes were sliced and attached into glutin-coated wells with endothelial cell surface down. After being cultured at a time-gradient interval from 48 to 6 h, the corneal slices were detached and reattached into new wells, respectively. Cells in the wells containing only a pure population of RCE cells were collected and cultured in 20% FBS-DMEM/F12 medium containing chondroitin sulfate, ocular extract, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), carboxymethyl-chitosan, N-acetylglucosamine hydrochloride, glucosamine hydrochloride, culture medium of rabbit corneal stromal cells and oxidation-degradation products of chondroitin sulfate at 37 degrees C, 5% CO(2). The cultured RCE cells, in quadrangle and polygonal shapes, proliferated to confluence 3 weeks later. During the subsequent subculture, the shape of RCE cells changed gradually from polygonal to more fibroblastic. A novel RCE cell line, growing at a steady rate, with a population doubling time of 53.8 h, has been established and subcultured to passage 67. Chromosome analysis showed that the RCE cells exhibited chromosomal aneuploidy with the modal chromosome number of 44. The results of immuno-cytochemical staining with neuron specific enolase (NSE) confirmed that the RCE cells were in neuroectodermal origin. Combined with the results of vascular endothelial growth factor (VEGF) treatment and endothelial cell morphology recovery, it can be concluded that the cell line established here is an RCE cell line. This RCE cell line may serve as a useful tool in theoretical researches of mammalian corneal endothelial cells, and may also have potential application in artificial corneal endothelium development.

  3. Establishment of a novel corneal endothelial cell line from domestic rabbit, Oryctolagus curiculus

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To develop a rabbit corneal endothelial (RCE) cell line, in vitro culture of RCE cells was initiated from Oryctolagus curiculus corneas and a novel RCE cell line was established in this study. To initiate the primary culture of RCE cells, corneas from rabbit eyes were sliced and attached into glutin-coated wells with endothelial cell surface down. After being cultured at a time-gradient interval from 48 to 6 h, the corneal slices were detached and reattached into new wells, respectively. Cells in the wells containing only a pure population of RCE cells were collected and cultured in 20% FBS-DMEM/F12 medium con- taining chondroitin sulfate, ocular extract, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), carboxymethyl-chitosan, N-acetylglucosamine hydrochloride, glucosamine hydrochloride, culture medium of rabbit corneal stromal cells and oxidation-degradation products of chondroitin sul- fate at 37℃, 5% CO2. The cultured RCE cells, in quadrangle and polygonal shapes, proliferated to con- fluence 3 weeks later. During the subsequent subculture, the shape of RCE cells changed gradually from polygonal to more fibroblastic. A novel RCE cell line, growing at a steady rate, with a population doubling time of 53.8 h, has been established and subcultured to passage 67. Chromosome analysis showed that the RCE cells exhibited chromosomal aneuploidy with the modal chromosome number of 44. The results of immuno-cytochemical staining with neuron specific enolase (NSE) confirmed that the RCE cells were in neuroectodermal origin. Combined with the results of vascular endothelial growth factor (VEGF) treatment and endothelial cell morphology recovery, it can be concluded that the cell line established here is an RCE cell line. This RCE cell line may serve as a useful tool in theoretical re- searches of mammalian corneal endothelial cells, and may also have potential application in artificial corneal endothelium development.

  4. Establishment of a novel corneal endothelial cell line from domestic rabbit, Oryctolagus curiculus

    Institute of Scientific and Technical Information of China (English)

    FAN TingJun; ZHAO Jun; FU YongFeng; CONG RiShan; GUO RuiChao; LIU WanShun; HAN BaoQin; YU QiuTao; WANG Jing

    2007-01-01

    To develop a rabbit corneal endothelial (RCE) cell line, in vitro culture of RCE cells was initiated from Oryctolagus curiculus corneas and a novel RCE cell line was established in this study. To initiate the primary culture of RCE cells, corneas from rabbit eyes were sliced and attached into glutin-coated wells with endothelial cell surface down. After being cultured at a time-gradient interval from 48 to 6 h, the corneal slices were detached and reattached into new wells, respectively. Cells in the wells containing only a pure population of RCE cells were collected and cultured in 20% FBS-DMEM/F12 medium containing chondroitin sulfate, ocular extract, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), carboxymethyl-chitosan, N-acetylglucosamine hydrochloride, glucosamine hydrochloride,culture medium of rabbit corneal stromal cells and oxidation-degradation products of chondroitin sulfate at 37℃, 5% CO2. The cultured RCE cells, in quadrangle and polygonal shapes, proliferated to confluence 3 weeks later. During the subsequent subculture, the shape of RCE cells changed gradually from polygonal to more fibroblastic. A novel RCE cell line, growing at a steady rate, with a population doubling time of 53.8 h, has been established and subcultured to passage 67. Chromosome analysis showed that the RCE cells exhibited chromosomal aneuploidy with the modal chromosome number of 44. The results of immuno-cytochemical staining with neuron specific enolase (NSE) confirmed that the RCE cells were in neuroectodermal origin. Combined with the results of vascular endothelial growth factor (VEGF) treatment and endothelial cell morphology recovery, it can be concluded that the cell line established here is an RCE cell line. This RCE cell line may serve as a useful tool in theoretical researches of mammalian corneal endothelial cells, and may also have potential application in artificial corneal endothelium development.

  5. Derivation of corneal endothelial cell-like cells from rat neural crest cells in vitro.

    Directory of Open Access Journals (Sweden)

    Chengqun Ju

    Full Text Available The aim of this study was to investigate the feasibility of inducing rat neural crest cells (NCC to differentiate to functional corneal endothelial cell (CEC-like cells in vitro. Rat NCC were induced with adult CEC-derived conditioned medium. Immunofluorescence, flow cytometry and real time RT-PCR assay were used to detect expression of the corneal endothelium differentiation marker N-cadherin and transcription factors FoxC1 and Pitx2. CFDA SE-labeled CEC-like cells were transplanted to the corneal endothelium of a rat corneal endothelium deficiency model, and an eye-down position was maintained for 24 hours to allow cell attachment. The animals were observed for as long as 2 months after surgery and underwent clinical and histological examination. Spindle-like NCC turned to polygonal CEC-like after induction and expressed N-cadherin, FoxC1, Pitx2, zonula occludens-1 and sodium-potassium pump Na(+/K(+ ATPase. The corneas of the experimental group were much clearer than those of the control group and the mean corneal thickness in the experimental group was significantly less than in the control group7, 14, 21 and 28 days after surgery. Confocal microscopy through focusing and histological analysis confirmed that green fluorescence-positive CEC-like cells formed a monolayer covering the Descemet's membrane in the experimental group. In conclusion, CEC-like cells derived from NCCs displayed characters of native CEC, and the induction protocol provides guidance for future human CEC induction from NCC.

  6. Effects of Phthalates on the Human Corneal Endothelial Cell Line B4G12

    DEFF Research Database (Denmark)

    Krüger, Tanja; Cao, Yi; Kjærgaard, Søren K.;

    2012-01-01

    Phthalates are industrial chemicals used in many cosmetics. We evaluated an in vitro model for eye irritancy testing using the human corneal endothelial cell line B4G12. Cell proliferation and toxicity were assessed after exposing to di-n-butyl phthalate (DBP), benzyl butyl phthalate (BBP), di-2...

  7. [The irido-corneo-endothelial syndrome. The loss of the control of corneal endothelial cell cycle. A review].

    Science.gov (United States)

    Robert, A M; Renard, G; Robert, L; Bourges, J-L

    2013-04-01

    The three major symptoms of the irido-corneo-endothelial syndrome are the alterations of the corneal endothelium and of the iris with a loss of the regulation of the cell cycle, and the progressive obstruction of the irido-corneal angle. This rare pathology attacks mainly young adult women. Most of the symptoms and complications originate from the excessive proliferation of the corneal endothelial cells accompanied by the evolution of their phenotype towards that of the epithelial cells. In normal conditions the corneal endothelial cells do not divide, they are blocked in the G1 stage of the cell cycle, mainly because of the action of the inhibitors of cyclin-dependent kinases. Still these cells retain a good capacity for proliferation, which can be induced by the down-regulation of the expression of the inhibitors of the cyclin-dependent kinases. This proliferative capacity declines with age and is also different according to the localization of the cells: it is more intense with those originating from the central area then in those from the peripheral area of the cornea. The age-related decline of the proliferative capacity is not due to the shortening of the telomers, but to the stress-induced accelerated senescence of the cells.

  8. Envisaging an allogenic Corneal endothelial precursor/Stem Cell Bank (CESBANK

    Directory of Open Access Journals (Sweden)

    Parikumar P

    2008-01-01

    Full Text Available Bullous Keratopathy (BK affects thousands of people in India every year. Though in early stages it is manageable medically, advanced disease warrants either total corneal transplantation or partial thickness transplantation for which a donor-cadaver cornea is necessary. Amano et al have reported the successful treatment of BK in animal models using in-vitro expanded human corneal endothelial precursors; though the rabbits had to be kept facing eye down to allow gravity assisted settling of the cells to the summit of the cornea where the damage had been created. For successful treatment using the above method, a human being has to lie prone with eyes immobilized for 24-36 Hrs. This is extremely discomforting and hence not practical. Corneal endothelium removed from the button and transported at varying temperature conditions for 48Hrs was successfully cultured in NCRM and this was reported earlier. We are working on a suitable scaffold to retain the cells in situ until their attachment to the damaged portion of the corneal endothelium enabling it to heal without the patient having to lie prone. With such capability, we envisage to make a corneal endothelial precursor/stem cell (CES bank named as CESBANK to make in-vitro expanded CES available for patients with corneal diseases, most commonly Bullous Keratopathy (BK.

  9. Inhibition of TGF-β signaling enables human corneal endothelial cell expansion in vitro for use in regenerative medicine.

    Directory of Open Access Journals (Sweden)

    Naoki Okumura

    Full Text Available Corneal endothelial dysfunctions occurring in patients with Fuchs' endothelial corneal dystrophy, pseudoexfoliation syndrome, corneal endotheliitis, and surgically induced corneal endothelial damage cause blindness due to the loss of endothelial function that maintains corneal transparency. Transplantation of cultivated corneal endothelial cells (CECs has been researched to repair endothelial dysfunction in animal models, though the in vitro expansion of human CECs (HCECs is a pivotal practical issue. In this study we established an optimum condition for the cultivation of HCECs. When exposed to culture conditions, both primate and human CECs showed two distinct phenotypes: contact-inhibited polygonal monolayer and fibroblastic phenotypes. The use of SB431542, a selective inhibitor of the transforming growth factor-beta (TGF-β receptor, counteracted the fibroblastic phenotypes to the normal contact-inhibited monolayer, and these polygonal cells maintained endothelial physiological functions. Expression of ZO-1 and Na(+/K(+-ATPase maintained their subcellular localization at the plasma membrane. Furthermore, expression of type I collagen and fibronectin was greatly reduced. This present study may prove to be the substantial protocol to provide the efficient in vitro expansion of HCECs with an inhibitor to the TGF-β receptor, and may ultimately provide clinicians with a new therapeutic modality in regenerative medicine for the treatment of corneal endothelial dysfunctions.

  10. Cultivation of Human Microvascular Endothelial Cells on Topographical Substrates to Mimic the Human Corneal Endothelium

    Directory of Open Access Journals (Sweden)

    Jie Shi Chua

    2013-03-01

    Full Text Available Human corneal endothelial cells have a limited ability to replicate in vivo and in vitro. Allograft transplantation becomes necessary when an accident or trauma results in excessive cell loss. The reconstruction of the cornea endothelium using autologous cell sources is a promising alternative option for therapeutic or in vitro drug testing applications. The native corneal endothelium rests on the Descemet’s membrane, which has nanotopographies of fibers and pores. The use of synthetic topographies mimics the native environment, and it is hypothesized that this can direct the behavior and growth of human microvascular endothelial cells (HMVECs to resemble the corneal endothelium. In this study, HMVECs are cultivated on substrates with micron and nano-scaled pillar and well topographies. Closely packed HMVEC monolayers with polygonal cells and well-developed tight junctions were formed on the topographical substrates. Sodium/potassium (Na+/K+ adenine triphosphatase (ATPase expression was enhanced on the microwells substrate, which also promotes microvilli formation, while more hexagonal-like cells are found on the micropillars samples. The data obtained suggests that the use of optimized surface patterning, in particular, the microtopographies, can induce HMVECs to adopt a more corneal endothelium-like morphology with similar barrier and pump functions. The mechanism involved in cell contact guidance by the specific topographical features will be of interest for future studies.

  11. Cultivation of corneal endothelial cells on a pericellular matrix prepared from human decidua-derived mesenchymal cells.

    Directory of Open Access Journals (Sweden)

    Ryohei Numata

    Full Text Available The barrier and pump functions of the corneal endothelium are essential for the maintenance of corneal transparency. Although corneal transplantation is the only current therapy for treating corneal endothelial dysfunction, the potential of tissue-engineering techniques to provide highly efficient and less invasive therapy in comparison to corneal transplantation has been highly anticipated. However, culturing human corneal endothelial cells (HCECs is technically difficult, and there is no established culture protocol. The aim of this study was to investigate the feasibility of using a pericellular matrix prepared from human decidua-derived mesenchymal cells (PCM-DM as an animal-free substrate for HCEC culture for future clinical applications. PCM-DM enhanced the adhesion of monkey CECs (MCECs via integrin, promoted cell proliferation, and suppressed apoptosis. The HCECs cultured on the PCM-DM showed a hexagonal morphology and a staining profile characteristic of Na⁺/K⁺-ATPase and ZO-1 at the plasma membrane in vivo, whereas the control HCECs showed a fibroblastic phenotype. The cell density of the cultured HCECs on the PCM-DM was significantly higher than that of the control cells. These results indicate that PCM-DM provides a feasible xeno-free matrix substrate and that it offers a viable in vitro expansion protocol for HCECs while maintaining cellular functions for use as a subsequent clinical intervention for tissue-engineered based therapy of corneal endothelial dysfunction.

  12. Cultivation of corneal endothelial cells on a pericellular matrix prepared from human decidua-derived mesenchymal cells.

    Science.gov (United States)

    Numata, Ryohei; Okumura, Naoki; Nakahara, Makiko; Ueno, Morio; Kinoshita, Shigeru; Kanematsu, Daisuke; Kanemura, Yonehiro; Sasai, Yoshiki; Koizumi, Noriko

    2014-01-01

    The barrier and pump functions of the corneal endothelium are essential for the maintenance of corneal transparency. Although corneal transplantation is the only current therapy for treating corneal endothelial dysfunction, the potential of tissue-engineering techniques to provide highly efficient and less invasive therapy in comparison to corneal transplantation has been highly anticipated. However, culturing human corneal endothelial cells (HCECs) is technically difficult, and there is no established culture protocol. The aim of this study was to investigate the feasibility of using a pericellular matrix prepared from human decidua-derived mesenchymal cells (PCM-DM) as an animal-free substrate for HCEC culture for future clinical applications. PCM-DM enhanced the adhesion of monkey CECs (MCECs) via integrin, promoted cell proliferation, and suppressed apoptosis. The HCECs cultured on the PCM-DM showed a hexagonal morphology and a staining profile characteristic of Na⁺/K⁺-ATPase and ZO-1 at the plasma membrane in vivo, whereas the control HCECs showed a fibroblastic phenotype. The cell density of the cultured HCECs on the PCM-DM was significantly higher than that of the control cells. These results indicate that PCM-DM provides a feasible xeno-free matrix substrate and that it offers a viable in vitro expansion protocol for HCECs while maintaining cellular functions for use as a subsequent clinical intervention for tissue-engineered based therapy of corneal endothelial dysfunction.

  13. Discovery of molecular markers to discriminate corneal endothelial cells in the human body.

    Directory of Open Access Journals (Sweden)

    Masahito Yoshihara

    Full Text Available The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant number of CECs results in corneal edema called bullous keratopathy which can lead to severe visual loss. Corneal transplantation is the most effective method to treat corneal endothelial dysfunction, where it suffers from donor shortage. Therefore, regeneration of CECs from other cell types attracts increasing interests, and specific markers of CECs are crucial to identify actual CECs. However, the currently used markers are far from satisfactory because of their non-specific expression in other cell types. Here, we explored molecular markers to discriminate CECs from other cell types in the human body by integrating the published RNA-seq data of CECs and the FANTOM5 atlas representing diverse range of cell types based on expression patterns. We identified five genes, CLRN1, MRGPRX3, HTR1D, GRIP1 and ZP4 as novel markers of CECs, and the specificities of these genes were successfully confirmed by independent experiments at both the RNA and protein levels. Notably none of them have been documented in the context of CEC function. These markers could be useful for the purification of actual CECs, and also available for the evaluation of the products derived from other cell types. Our results demonstrate an effective approach to identify molecular markers for CECs and open the door for the regeneration of CECs in vitro.

  14. Discovery of molecular markers to discriminate corneal endothelial cells in the human body.

    Science.gov (United States)

    Yoshihara, Masahito; Ohmiya, Hiroko; Hara, Susumu; Kawasaki, Satoshi; Hayashizaki, Yoshihide; Itoh, Masayoshi; Kawaji, Hideya; Tsujikawa, Motokazu; Nishida, Kohji

    2015-01-01

    The corneal endothelium is a monolayer of hexagonal corneal endothelial cells (CECs) on the inner surface of the cornea. CECs are critical in maintaining corneal transparency through their barrier and pump functions. CECs in vivo have a limited capacity in proliferation, and loss of a significant number of CECs results in corneal edema called bullous keratopathy which can lead to severe visual loss. Corneal transplantation is the most effective method to treat corneal endothelial dysfunction, where it suffers from donor shortage. Therefore, regeneration of CECs from other cell types attracts increasing interests, and specific markers of CECs are crucial to identify actual CECs. However, the currently used markers are far from satisfactory because of their non-specific expression in other cell types. Here, we explored molecular markers to discriminate CECs from other cell types in the human body by integrating the published RNA-seq data of CECs and the FANTOM5 atlas representing diverse range of cell types based on expression patterns. We identified five genes, CLRN1, MRGPRX3, HTR1D, GRIP1 and ZP4 as novel markers of CECs, and the specificities of these genes were successfully confirmed by independent experiments at both the RNA and protein levels. Notably none of them have been documented in the context of CEC function. These markers could be useful for the purification of actual CECs, and also available for the evaluation of the products derived from other cell types. Our results demonstrate an effective approach to identify molecular markers for CECs and open the door for the regeneration of CECs in vitro.

  15. Impact of temporary hyperthermia on corneal endothelial cell survival during organ culture preservation.

    Science.gov (United States)

    Schroeter, Jan; Ruggeri, Alfredo; Thieme, Hagen; Meltendorf, Christian

    2015-05-01

    To evaluate temporary exposure to hyperthermia for its impact on endothelial cell density of porcine corneas in organ culture medium containing dextran with regards to possible negative influences of high temperatures during the storage and transport of corneal grafts. Four groups of central discs (diameter 8 mm) from the corneas of both eyes in 40 pigs were first organ-cultured (MEM with 6% dextran 500) for 24 h at 32°C. Ten corneas were then exposed to 40°C in group 1, to 42°C in group 2, to 44°C in group 3, and to 50°C in group 4 for 12 h each. The paired corneal discs for all groups were not treated, stored at 32°C and served as controls. After further organ culture of all corneas for 48 h at 32°C to allow regenerative processes, corneal endothelium was stained with Alizarin Red S and examined by light microscopy. The endothelial cell densities were determined on three central images using a system for the automatic estimation of morphometric parameters of corneal endothelium. Exposure for 12 h to 40°C as well as to 42°C induced no endothelial cell loss. Statistical analysis showed no significant difference of the endothelial cell density between corneas exposed to 40°C and 42°C and the control corneas (40°C treatment: 4736 ± 426 cells/mm(2) and control: 4762 ± 344 cells/mm(2), p = 0.74; 42°C treatment: 4240 ± 363 cells/mm(2) and control: 4176 ± 448 cells/mm(2), p = 0.40). Exposure to 44°C and 50°C lead to total necrosis of the endothelial cell layer. Exposure of organ cultured porcine corneas in dextran containing medium up to 42°C for 12 h does not compromise the endothelial cell density in a clinically relevant manner. Temperatures above 42°C, as it might be the case during transports from the cornea bank to the ophthalmic surgeon, must be strictly avoided as they damage the endothelial cell layer.

  16. Mitochondrial and Morphologic Alterations in Native Human Corneal Endothelial Cells Associated With Diabetes Mellitus.

    Science.gov (United States)

    Aldrich, Benjamin T; Schlötzer-Schrehardt, Ursula; Skeie, Jessica M; Burckart, Kimberlee A; Schmidt, Gregory A; Reed, Cynthia R; Zimmerman, M Bridget; Kruse, Friedrich E; Greiner, Mark A

    2017-04-01

    To characterize changes in the energy-producing metabolic activity and morphologic ultrastructure of corneal endothelial cells associated with diabetes mellitus. Transplant suitable corneoscleral tissue was obtained from donors aged 50 to 75 years. We assayed 3-mm punches of endothelium-Descemet membrane for mitochondrial respiration and glycolysis activity using extracellular flux analysis of oxygen and pH, respectively. Transmission electron microscopy was used to assess qualitative and quantitative ultrastructural changes in corneal endothelial cells and associated Descemet membrane. For purposes of analysis, samples were divided into four groups based on a medical history of diabetes regardless of type: (1) nondiabetic, (2) noninsulin-dependent diabetic, (3) insulin-dependent diabetic, and (4) insulin-dependent diabetic with specified complications due to diabetes (advanced diabetic). In total, 229 corneas from 159 donors were analyzed. Insulin-dependent diabetic samples with complications due to diabetes displayed the lowest spare respiratory values compared to all other groups (P ≤ 0.002). The remaining mitochondrial respiration and glycolysis metrics did not differ significantly among groups. Compared to nondiabetic controls, the endothelium from advanced diabetic samples had alterations in mitochondrial morphology, pronounced Golgi bodies associated with abundant vesicles, accumulation of lysosomal bodies/autophagosomes, and focal production of abnormal long-spacing collagen. Extracellular flux analysis suggests that corneal endothelial cells of donors with advanced diabetes have impaired mitochondrial function. Metabolic findings are supported by observed differences in mitochondrial morphology of advanced diabetic samples but not controls. Additional studies are needed to determine the precise mechanism(s) by which mitochondria become impaired in diabetic corneal endothelial cells.

  17. Genetics of corneal endothelial dystrophies

    Indian Academy of Sciences (India)

    Chitra Kannabiran

    2009-12-01

    The corneal endothelium maintains the level of hydration in the cornea. Dysfunction of the endothelium results in excess accumulation of water in the corneal stroma, leading to swelling of the stroma and loss of transparency. There are four different corneal endothelial dystrophies that are hereditary, progressive, non-inflammatory disorders involving dysfunction of the corneal endothelium. Each of the endothelial dystrophies is genetically heterogeneous with different modes of transmission and/or different genes involved in each subtype. Genes responsible for disease have been identified for only a subset of corneal endothelial dystrophies. Knowledge of genes involved and their function in the corneal endothelium can aid understanding the pathogenesis of the disorder as well as reveal pathways that are important for normal functioning of the endothelium.

  18. Serial explant culture provides novel insights into the potential location and phenotype of corneal endothelial progenitor cells.

    Science.gov (United States)

    Walshe, Jennifer; Harkin, Damien G

    2014-10-01

    The routine cultivation of human corneal endothelial cells, with the view to treating patients with endothelial dysfunction, remains a challenging task. While progress in this field has been buoyed by the proposed existence of progenitor cells for the corneal endothelium at the corneal limbus, strategies for exploiting this concept remain unclear. In the course of evaluating methods for growing corneal endothelial cells, we have noted a case where remarkable growth was achieved using a serial explant culture technique. Over the course of 7 months, a single explant of corneal endothelium, acquired from cadaveric human tissue, was sequentially seeded into 7 culture plates and on each occasion produced a confluent cell monolayer. Sample cultures were confirmed as endothelial in origin by positive staining for glypican-4. On each occasion, small cells, closest to the tissue explant, developed into a highly compact layer with an almost homogenous structure. This layer was resistant to removal with trypsin and produced continuous cell outgrowth during multiple culture periods. The small cells gave rise to larger cells with phase-bright cell boundaries and prominent immunostaining for both nestin and telomerase. Nestin and telomerase were also strongly expressed in small cells immediately adjacent to the wound site, following transfer of the explant to another culture plate. These findings are consistent with the theory that progenitor cells for the corneal endothelium reside within the limbus and provide new insights into expected expression patterns for nestin and telomerase within the differentiation pathway.

  19. Pterygium is related to a decrease in corneal endothelial cell density.

    Science.gov (United States)

    Hsu, Min-Yen; Lee, Hsin-Nung; Liang, Chiao-Ying; Wei, Li-Chen; Wang, Chun-Yuan; Lin, Keng-Hung; Shen, Ying-Cheng

    2014-07-01

    The aim of this study was to investigate the relationship between pterygium and a decrease in the corneal endothelial cell density (ECD) in patients with unilateral primary pterygium. In this retrospective cross-sectional study, 90 consecutive patients with unilateral primary pterygium were enrolled from January 2010 to June 2012. Corneal ECD was measured in both eyes, and the fellow eyes were considered as controls. The relationship between the percentage of pterygium to cornea and a decrease in the ECD was analyzed. An increase in astigmatism in eyes with pterygium was evaluated for association with decreased ECD using the Pearson correlation test. The percentage of pterygium to cornea ranged from 3.5% to 65.2%, with a median of 12.35%. The difference in the corneal ECD between eyes with pterygium and control eyes ranged from +9.6% to -37.7%, with a median of -9.75%. The results of the Pearson correlation statistical test showed a strong logarithmic correlation between a decrease in the corneal ECD and the percentage of pterygium to cornea (R = 0.688, P decrease in the ECD in eyes with pterygium. Pterygium is related to a decrease in corneal ECD. Surgical intervention should be considered in patients with extensive pterygium involvement in the cornea or a significant increase in astigmatism.

  20. Corneal endothelial cell loss during phacoemulsification: bevel-up versus bevel-down phaco tip.

    Science.gov (United States)

    Faramarzi, Amir; Javadi, Mohammad Ali; Karimian, Farid; Jafarinasab, Mohammad Reza; Baradaran-Rafii, Alireza; Jafari, Fariba; Yaseri, Mehdi

    2011-11-01

    To compare corneal endothelial cell loss during cataract extraction by phacoemulsification with 2 different phaco-tip positions. Ophthalmic Research Center and Department of Ophthalmology, Labbafinejad Medical Center, Shahid Beheshti Medical University, Tehran, Iran. Randomized clinical trial. Eyes scheduled for cataract extraction were randomly assigned stop-and-chop phacoemulsification with the phaco tip in the conventional bevel-up position or with the phaco tip in the bevel-down position. During surgery, the effective phacoemulsification time (EPT) was recorded. Preoperative endothelial cell parameters were compared with measurements taken 3 months postoperatively. Each group comprised 30 eyes (30 patients). There were no statistically significant differences in age, sex, anterior chamber depth, axial length, or EPT between the 2 groups. The mean preoperative endothelial cell density (ECD) was 2544 cells/mm(2) ± 64 (SD) in the bevel-up group and 2471 ± 59 cells/mm(2) in the bevel-down group (P=.610). Postoperatively, both groups had a significant decrease in ECD. The mean endothelial cell loss was 5.9% in the bevel-up group and 13.6% in the bevel-down group (P=.012). The percentage of hexagonal cells and coefficient of variation in cell size were not different between the 2 groups preoperatively or postoperatively; however, after surgery, there was a significant decrease in the percentage of hexagonal cells in both groups. Corneal endothelial cell loss during phacoemulsification was significantly higher when the phaco tip was in the bevel-down position than in the conventional bevel-up position. No author has a financial or proprietary interest in any material or method mentioned. Copyright © 2011 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

  1. Cytotoxic effects of betaxolol on healthy corneal endothelial cells both in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Ying Miao

    2014-02-01

    Full Text Available AIM: To demonstrate the cytotoxic effect of betaxolol and its underlying mechanism on human corneal endothelial cells(HCE cells in vitro and cat corneal endothelial cells(CCE cells in vivo, providing experimental basis for safety anti-glaucoma drug usage in clinic of ophthalmology.METHODS: In vivo and in vitro experiments were conducted to explore whether and how betaxolol participates in corneal endothelial cell injury. The in vitro morphology, growth status, plasma membrane permeability, DNA fragmentation, and ultrastructure of HCE cells treated with 0.021875-0.28g/L betaxolol were examined by light microscope, 3-(4,5-dimethylthiahiazo (-z-y1-3,5-di-phenytetrazoliumromide (MTT assay, acridine orange (AO/ethidium bromide (EB double-fluorescent staining, DNA agarose gel electrophoresis, and transmission electron microscope (TEM. The in vivo density, morphology, and ultrastructure of CCE cells, corneal thickness, and eye pressure of cat eyes treated with 0.28g/L betaxolol were investigated by specular microscopy, applanation tonometer, alizarin red staining, scanning electron microscope (SEM, and TEM.RESULTS: Exposure to betaxolol at doses from 0.0875g/L to 2.8g/L induced morphological and ultrastructural changes of in vitro cultured HCE cells such as cytoplasmic vacuolation, cellular shrinkage, structural disorganization, chromatin condensation, and apoptotic body appearance. Simultaneously, betaxolol elevated plasma membrane permeability and induced DNA fragmentation of these cells in a dose-dependent manner in AO/EB staining. Furthermore, betaxolol at a dose of 2.8g/L also induced decrease of density of CCE cells in vivo, and non-hexagonal and shrunk apoptotic cells were also found in betaxolol-treated cat corneal endothelia.CONCLUSION: Betaxolol has significant cytotoxicity on HCE cells in vitro by inducing apoptosis of these cells, and induced apoptosis of CCE cells in vivo as well. The findings help provide new insight into the apoptosis

  2. In vitro evaluation of the interactions between human corneal endothelial cells and extracellular matrix proteins.

    Science.gov (United States)

    Choi, Jin San; Kim, Eun Young; Kim, Min Jeong; Giegengack, Matthew; Khan, Faraaz A; Khang, Gilson; Soker, Shay

    2013-02-01

    The corneal endothelium is the innermost cell layer of the cornea and rests on Descemet's membrane consisting of various extracellular matrix (ECM) proteins which can directly affect the cellular behaviors such as cell adhesion, proliferation, polarity, morphogenesis and function. The objective of this study was to investigate the interactions between the ECM environment and human corneal endothelial cells (HCECs), with the ultimate goal to improve cell proliferation and function in vitro. To evaluate the interaction of HCECs with ECM proteins, cells were seeded on ECM-coated tissue culture dishes, including collagen type I (COL I), collagen type IV (COL IV), fibronectin (FN), FNC coating mix (FNC) and laminin (LM). Cell adhesion and proliferation of HCECs on each substratum and expression of CEC markers were studied. The results showed that HCECs plated on the COL I, COL IV, FN and FNC-coated plates had enhanced cell adhesion initially; the number for COL I, COL IV, FN and FNC was significantly higher than the control (P < 0.05). In addition, cells grown on ECM protein-coated dishes showed more compact cellular morphology and CEC marker expression compared to cells seeded on uncoated dishes. Collectively, our results suggest that an adequate ECM protein combination can provide a long-term culture environment for HCECs for corneal endothelium transplantation.

  3. Isolation and transplantation of corneal endothelial cell-like cells derived from in-vitro-differentiated human embryonic stem cells.

    Science.gov (United States)

    Zhang, Kai; Pang, Kunpeng; Wu, Xinyi

    2014-06-15

    The maintenance of corneal dehydration and transparency depends on barrier and pump functions of corneal endothelial cells (CECs). The human CECs have no proliferation capacity in vivo and the ability to divide in vitro under culture conditions is dramatically limited. Thus, the acquisition of massive cells analogous to normal human CECs is extremely necessary whether from the perspective of cellular basic research or from clinical applications. Here we report the derivation of CEC-like cells from human embryonic stem cells (hESCs) through the periocular mesenchymal precursor (POMP) phase. Using the transwell coculture system of hESCs with differentiated human corneal stromal cells, we induced hESCs to differentiate into POMPs. Then, CEC-like cells were derived from POMPs with lens epithelial cell-conditioned medium. Within 1 week, CEC-like cells that expressed the corneal endothelium (CE) differentiation marker N-cadherin and transcription factors FoxC1 and Pitx2 were detectable. Fluorescence-activated cell sorting (FACS)-based isolation of the N-cadherin/vimentin dual-positive population enriches for CEC-like cells. The isolated CEC-like cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) and seeded onto posterior acellular porcine corneal matrix lamellae to construct the CEC-like cell sheets. Pump function parameters of the CEC-like cell sheets approximated those of human donor corneas. Importantly, when the CEC-like cell sheets were transplanted into the eyes of rabbit CE dysfunction models, the corneal transparency was restored gradually. In conclusion, CEC-like cells derived from hESCs displayed characteristics of native human CECs. This renewable source of human CECs offers massive cells for further studies of human CEC biological characteristics and potential applications of replacement therapies as substitution for donor CECs in the future.

  4. [Comparison of corneal endothelial cells after ECCE and phacoemulsification of the lens].

    Science.gov (United States)

    Trnavec, B; Cuvala, J; Cernák, A; Vodrázková, E

    1997-08-01

    The authors evaluate the finding on the corneal endothelium before and after operation of cataract. For examination of the endothelium they used a specular microscope SP 1.000 of Topcon Co. The group comprised 64 eyes of 64 patients. The patients were divided at random into two groups. The first group comprised 26 patients, where ECCE was performed. The second group comprised 38 patients and the opaque lens was removed by phacoemulsification. The mean age in the first group was 69.3 years and in the second group 70.7 years. The same viscoelastic material (methylcellulose) was used and the same synthetic lens from PMMA material was implanted into the capsule. The endothelium was examined one day after operation and on the 7th to 10th day after operation. The following parameters were evaluated: density of the endothelial cells in the centre, mean cell size, polymegethism, coefficient of variation, pleomorphism. In the group of patients who had ECCE the loss of endothelial cells was 18.53%, in the group with phacoemulsification of the nucleus the loss was 16.43%. This difference is not statistically significant. After operation in both groups enlargement of the minimal, maximal as well as mean cell size was observed, the coefficient of variation increased while the grade of cell hexagonality decreased. However these differences in endothelial cells were not statistically significant. After operation of cataract not only endothelial cells are lost but also significant changes in cell morphology occur.

  5. Correlation between Corneal Endothelial Cell Loss and Location of Phacoemulsification Incision

    Directory of Open Access Journals (Sweden)

    Hamid Gharaee

    2011-01-01

    Full Text Available Purpose: To assess the relationship between corneal endothelial cell loss after phacoemulsification and the location of the clear corneal incision. Methods: A total of 92 patients (92 eyes with senile cataracts who met the study criteria were included in this cross sectional study and underwent phacoemulsification. The incision site was determined based on the steep corneal meridian according to preoperative keratometry. Endothelial cell density was measured using specular microscopy in the center and 3 mm from the center of the cornea in the meridian of the incisions (temporal, superior, and superotemporal. Phacoemulsification was performed by a single surgeon using the phaco chop technique through a 3.2 mm clear cornea incision. Endothelial cell loss (ECL was evaluated 1 week, and 1 and 3 months postoperatively. Results: At all time points during follow-up, ECL was comparable among the 3 incision sites, both in the central cornea and in the meridian of the incision (P > 0.05 for all comparisons. However, 3 months postoperatively, mean central ECL with superior incisions and mean sectoral ECL with temporal incisions were slightly higher. Superotemporal incisions entailed slightly less ECL than the other 2 groups. Overall, one month after surgery, mean central ECL was 10.8% and mean ECL in the sector of the incisions was 14.0%. Axial length and effective phaco time (EFT were independent predictors of postoperative central ECL (P values 0.005 and < 0.0001, respectively. Conclusion: A superotemporal phacoemulsification incision may entail less ECL as compared to other incisions (although not significantly different. The amount of central ECL may be less marked in patients with longer axial lengths and with procedures utilizing less EFT.

  6. Corneal Endothelial Cell Changes Due to Combined Phacoemulsification-Posterior Chamber Intraocular Lens Implantation and Transpupillary Silicone Oil Removal

    Directory of Open Access Journals (Sweden)

    Mehmet Hanifi Alp

    2014-12-01

    Full Text Available Objectives: The aim of this study was to evaluate the effects of combined phacoemulsification-posterior chamber intraocular lens (PCIOL implantation and removal of transpupillary silicone oil on the corneal endothelial cell layer. Materials and Methods: In this study, we included seven eyes of 7 patients who had intravitreal hemorrhage resulting from retinal detachment or tractional retinal detachment due to proliferative diabetic retinopathy and who underwent pars plana vitrectomy and silicone oil injection. Complicated cataract developed, and phacoemulsification-PC-IOL implantation combined with transpupillary silicon oil removal were performed. Preoperative and postoperative corneal endothelial cell count values were taken by Topcon SP-2000P automatic non-contact specular microscopy and were assessed by IMAGEnet 2000 Endothelial Cell Analysis. The results were compared with the findings in the literature. Results: The mean endothelial cell density (ECD was 2461 cell/mm2 preoperatively. The silicone oil removal was performed after an average of 10±3.5 months (range 5-14 months, and then endothelial cells were counted after a mean of 17±21.2 months (range 1-49 months of the combined surgery. The mean ECD was determined as 1906 cell/mm2 postoperatively. While mean endothelial cell loss was found to be 32% in two patients with diabetes mellitus, five non-diabetic patients had 19% cell loss. Corneal decompensation was not observed in any patient during the follow-up period. Conclusion: After combined phacoemulsification-PC-IOL implantation and transpupillary silicone oil removal, significant reduction in endothelial cells was observed. We detected that this reduction was greater in diabetics. (Turk J Ophthalmol 2014; 44: 424-7

  7. Ex vivo expansion of bovine corneal endothelial cells in xeno-free medium supplemented with platelet releasate.

    Science.gov (United States)

    Chou, Ming-Li; Burnouf, Thierry; Wang, Tsung-Jen

    2014-01-01

    Clinical-grade ex vivo expansion of corneal endothelial cells can increase the availability of corneal tissues for transplantation and treatment of corneal blindness. However, these cells have very limited proliferative capacity. Successful propagation has required so far to use very complex growth media supplemented with fetal bovine serum and other xenocomponents. We hypothesized that human platelet releasates rich in multiple growth factors, and in particular neurotrophins, could potentially be a useful supplement for ex vivo expansion of corneal endothelium cells due to their neural crest origin. Platelet releasates were prepared by calcium salt activation of apheresis platelet concentrates, subjected or not to complement inactivation by heat treatment at 56°C for 30 minutes. Platelet releasates were characterized for their content in proteins and were found to contain high amount of growth factors including platelet-derived growth factor-AB (30.56 to 39.08 ng/ml) and brain-derived neurotrophic factor (30.57 to 37.11 ng/ml) neurotrophins. We compared the growth and viability of corneal endothelium cells in DMEM-F12 medium supplemented with different combinations of components, including 2.5%∼10% of the platelet releasates. Corneal endothelium cells expanded in platelet releasates exhibited good adhesion and a typical hexagonal morphology. Their growth and viability were enhanced when using the complement-inactivated platelet releasate at a concentration of 10%. Immunostaining and Western blots showed that CECs maintained the expressions of four important membrane markers: Na-K ATPase α1, zona occludens-1, phospho-connexin 43 and N-cadherin. In conclusion, our study provides the first proof-of-concept that human platelet releasates can be used for ex vivo expansion of corneal endothelium cells. These findings open a new paradigm for ex vivo propagation protocols of corneal endothelium cells in compliance with good tissue culture practices and regulatory

  8. Ex vivo expansion of bovine corneal endothelial cells in xeno-free medium supplemented with platelet releasate.

    Directory of Open Access Journals (Sweden)

    Ming-Li Chou

    Full Text Available Clinical-grade ex vivo expansion of corneal endothelial cells can increase the availability of corneal tissues for transplantation and treatment of corneal blindness. However, these cells have very limited proliferative capacity. Successful propagation has required so far to use very complex growth media supplemented with fetal bovine serum and other xenocomponents. We hypothesized that human platelet releasates rich in multiple growth factors, and in particular neurotrophins, could potentially be a useful supplement for ex vivo expansion of corneal endothelium cells due to their neural crest origin. Platelet releasates were prepared by calcium salt activation of apheresis platelet concentrates, subjected or not to complement inactivation by heat treatment at 56°C for 30 minutes. Platelet releasates were characterized for their content in proteins and were found to contain high amount of growth factors including platelet-derived growth factor-AB (30.56 to 39.08 ng/ml and brain-derived neurotrophic factor (30.57 to 37.11 ng/ml neurotrophins. We compared the growth and viability of corneal endothelium cells in DMEM-F12 medium supplemented with different combinations of components, including 2.5%∼10% of the platelet releasates. Corneal endothelium cells expanded in platelet releasates exhibited good adhesion and a typical hexagonal morphology. Their growth and viability were enhanced when using the complement-inactivated platelet releasate at a concentration of 10%. Immunostaining and Western blots showed that CECs maintained the expressions of four important membrane markers: Na-K ATPase α1, zona occludens-1, phospho-connexin 43 and N-cadherin. In conclusion, our study provides the first proof-of-concept that human platelet releasates can be used for ex vivo expansion of corneal endothelium cells. These findings open a new paradigm for ex vivo propagation protocols of corneal endothelium cells in compliance with good tissue culture practices

  9. Transforming growth factor-β2 induces morphological alteration of human corneal endothelial cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Jing; Wang; Ting-Jun; Fan; Xiu-Xia; Yang; Shi-Min; Chang

    2014-01-01

    AIM:To investigate the morphological altering effect of transforming growth factor-β2(TGF-β2) on untransfected human corneal endothelial cells(HCECs)in vitro.METHODS:After untransfected HCECs were treated with TGF-β2 at different concentrations, the morphology,cytoskeleton distribution, and type IV collagen expression of the cells were examined with inverted contrast light microscopy, fluorescence microscopy,immunofluorescence or Western Blot.RESULTS:TGF-β2 at the concentration of 3-15 μg/L had obviously alterative effects on HCECs morphology in dose and time-dependent manner, and 9 μg/L was the peak concentration. TGF-β2(9 μg/L) altered HCE cell morphology after treatment for 36 h, increased the mean optical density(P <0.01) and the length of F-actin,reduced the mean optical density(P <0.01) of the collagen type IV in extracellular matrix(ECM) and induced the rearrangement of F-actin, microtubule in cytoplasm and collagen type IV in ECM after treatment for 72 h.·CONCLUTION: TGF-β2 has obviously alterative effect on the morphology of HCECs from polygonal phenotype to enlarged spindle-shaped phenotype, in dose and time-dependence manner by inducing more, elongation and alignment of F-actin, rearrangement of microtubule and larger spread area of collagen type IV.

  10. A comparison of three methods for trephining donor corneal buttons: endothelial cell loss and microscopic ultrastructural evaluation.

    Science.gov (United States)

    Moshirfar, Majid; Meyer, Jay J; Kang, Paul C

    2009-11-01

    To evaluate the ultrastructure of the cut edge and associated endothelial cell loss following donor cornea trephination with a standard punch, vacuum punch, and vacuum trephine and artificial anterior chamber system. This laboratory investigation compared trephinations (8.0 mm) performed on human corneas using either a standard posterior punch (n = 12), vacuum posterior punch (n = 12), or vacuum trephine and artificial anterior chamber system (n = 12). Specular microscopy was performed before and after trephination to determine central endothelial cell density. Light and scanning electron microscopy were performed to evaluate the structure of the trephined edge. Endothelial cell-free distances from the trephinated edges were measured on light microscopy sections. Central endothelial cell loss (cells/mm(2)) after trephination was -14.0 +/- 49.9 (SD) for the standard posterior punch, -85.6 +/- 87.0 for the vacuum posterior punch, -116.0 +/- 223.1 for the vacuum trephine and artificial anterior chamber system. Endothelial cell-free distances from the trephined margin were 63 +/- 22 microm, 85 +/- 13 microm, and 123 +/- 48 microm for the three respective methods. The edges of grafts cut with anterior trephination were inward sloping from the epithelial to endothelial surfaces, while both posterior punches created outward sloping edges. Increased fibrillar disruption at edges was seen following anterior trephination. Different trephination methods produce distinct cut morphologies with the anterior trephination approach, resulting in more irregular margins. The anterior approach was associated with increased variability and greater endothelial cell loss than the studied posterior approaches. The use of corneal scissors may contribute to the morphologic features of the corneal button seen following anterior trephination.

  11. PDI-mediated ER retention and proteasomal degradation of procollagen I in corneal endothelial cells.

    Science.gov (United States)

    Ko, MinHee K; Kay, EunDuck P

    2004-04-15

    Procollagen I in corneal endothelial cells (CECs) is intracellularly degraded immediately after its synthesis. In this study, we investigated the mechanism of intracellular degradation of procollagen I by determining the role of protein disulfide isomerase (PDI) in endoplasmic reticulum (ER) retention and further determined the degradation pathway of procollagen I in CECs. When association of PDI to monomeric proalpha chains or the trimeric procollagen I carboxyl propeptides (PICPs) was analyzed, immune complex precipitated with anti-PICP antibody contained more PDI than that precipitated with antibodies to monomeric chains. PICPs were completely colocalized with PDI. When CECs were transfected with PDI vector, procollagen I and the recombinant PDI were colocalized in the ER, whereas CECs transfected with PDI minus KDEL (the ER retrieval sequence) vector demonstrated that the two proteins were localized in the Golgi and were subsequently secreted into the medium. Ribostamycin (an inhibitor of the chaperone activity of PDI) blocked colocalization of PDI and procollagen I. Cells treated with chloroquine (lysosome inhibitor) did not alter the subcellular localization of procollagen I, because the inhibitor failed to induce the accumulation of procollagen I at Golgi. On the other hand, procollagen I was colocalized with ubiquitin in the cytoplasm, and proteasomal inhibitors further facilitated the colocalization of the two proteins and accumulation of ubiquitinated procollagen I ladders. These results suggest that association of PDI with procollagen I, whether monomeric or trimeric, leads to ER retention of procollagen I before intracellular degradation via the ubiquitin-proteasome pathway.

  12. Involvement of P38MAPK in human corneal endothelial cell migration induced by TGF-β(2).

    Science.gov (United States)

    Joko, Takeshi; Shiraishi, Atsushi; Akune, Yoko; Tokumaru, Sho; Kobayashi, Takeshi; Miyata, Kazunori; Ohashi, Yuichi

    2013-03-01

    Because human corneal endothelial cells do not proliferate once the endothelial monolayer is formed, corneal wound healing is thought to be mediated by cell enlargement or migration rather than proliferation. However, the cellular mechanisms involved in corneal wound healing have not been fully determined. Because transforming growth factor-β(2) (TGF-β(2)) isoform is present in high concentrations in normal human aqueous humor, it may play a role in human corneal endothelial cell wound healing. The purpose of this study was to determine the effect of TGF-β(2) on the proliferation and migration of cultured human corneal endothelial cells (HCECs). To achieve this, we first examined the effect of TGF-β(2) on the wound closure rate in an in vitro HCEC wound healing model. However, unexpectedly TGF-β(2) had no effect on the wound closure rate in this model. Therefore, a real-time cell electronic sensing (RT-CES) system and the BrdU incorporation assay were used to determine the effect of TGF-β(2) (0.1-10 ng/ml) on cultured HCEC proliferation during in vitro wound healing. The specificity of this effect was confirmed by adding the TGF-β receptor I kinase inhibitor. TGF-β(2) inhibited the proliferation of HCECs in a dose dependent way and was blocked by TGF-β receptor I kinase inhibitor. Next, the Boyden chamber assay was used to determine how TGF-β(2) (10 ng/ml) affect HCEC migration. Exposure to TGF-β(2) increased cell migration, and a synergistic effect was observed when FGF-2 was added. To determine whether the mitogen-activated protein kinase (MAPK) signaling pathway is involved in the migration of HCECs, western blot analysis and Bio-Plex™ suspension array were used to detect phosphorylation of Erk1/2, p38, and JNK in HCECs stimulated by TGF-β(2) and/or FGF-2. The effect of the p38 MAPK inhibitor, SB239063 (10 μM), on TGF-β(2) and/or FGF-2-induced cellular migration was determined by the Boyden chamber assay. Both TGF-β(2) and FGF-2-induced p38

  13. Evaluation of the viability of cultured corneal endothelial cells by quantitative electron probe X-ray microanalysis.

    Science.gov (United States)

    Alaminos, M; Sanchez-Quevedo, M C; Muñoz-Avila, J I; García, J M; Crespo, P V; González-Andrades, M; Campos, A

    2007-06-01

    Construction of artificial organs and tissues by tissue engineering is strongly dependent on the availability of viable cells. For that reason, the viability and the physiological status of cells kept in culture must be evaluated before the cells can be used for clinical purposes. In this work, we determined the viability of isolated rabbit corneal endothelial cells by trypan blue staining and quantitative electron probe X-ray microanalysis. Our results showed that the ionic content of potassium in cultured corneal endothelial cells tended to rise initially, but significantly decreased in cells in the fifth (and final) subculture, especially in comparison to cells in the fourth subculture (P subculture than in the fourth subculture (P subculture (P = 0.031). These data imply a remarkable decrease in the K/Na ratio from the fourth to the fifth subculture. Our microanalytical results, along with the morphological differences between cells in the last two subcultures, are compatible with an early phase of the preapoptotic process in the fifth subculture, and suggest that cells of the first four subcultures would be better candidates for tissue engineering.

  14. Toxicity of antiglaucoma drugs with and without benzalkonium chloride to cultured human corneal endothelial cells

    Directory of Open Access Journals (Sweden)

    Masahiko Ayaki

    2010-10-01

    Full Text Available Masahiko Ayaki1, Atsuo Iwasawa2, Yoichi Inoue31Department of Ophthalmology, Saitama National Hospital, Wako, Japan; 2Life Particle Interaction Engineering Creation, New Industry Creation Hatchery Center, Tohoku University, Sendai, Japan; 3Department of Ophthalmology, Olympia Eye Hospital, Tokyo, JapanPurpose: The toxicity of antiglaucoma medications to ocular surface cells has been evaluated extensively; however, the toxicity to corneal endothelial cells (CECs remains elusive. Our aim is to evaluate the toxicity of antiglaucoma medications to CECs using an in vitro toxicity assay.Methods: Primary cultures of human (H CECs derived from eye bank specimens were established. Following exposure of HCECs to test solutions for 10, 30, or 60 minutes, or 48 hours, we measured cell viability using a WST-1 assay. Test solutions were diluted in culture media and included 0.5% Timoptol®, preservative-free 0.5% timolol maleate, 1% Trusopt®, preservative-free 1% dorzolamide, Travatan®, Travatan Z®, Xalatan®, and benzalkonium chloride (BAK. To assess cell viability, the value of the test culture well after treatment was expressed as a percentage of that of the control well. Toxicity of each solution was compared using the cell viability score (CVS.Results: After exposure to 10-fold dilutions of test solutions for 48 hours, HCEC viabilities were 48.5% for 0.5% Timoptol, 80.9% for preservative-free 0.5% timolol maleate, 47.0% for 1% Trusopt, 71.7% for preservative-free 1% dorzolamide, 55.5% for Travatan, 88.5% for Travatan Z, and 52.5% for Xalatan. Exposure to test solutions diluted 100-fold or more resulted in HCEC viabilities > 80%, with the exception of preservative-free 1% dorzolamide, which resulted in a viability of 72.0% at a dilution of 100-fold. Based on CVS, the order of cell viability was Travatan Z ≥ preservative-free timolol maleate = preservative-free dorzolamide > 0.5% Timoptol = 1% Trusopt > Travatan ≥ Xalatan. Assessment of the

  15. Toxicity of antiglaucoma drugs with and without benzalkonium chloride to cultured human corneal endothelial cells

    Science.gov (United States)

    Ayaki, Masahiko; Iwasawa, Atsuo; Inoue, Yoichi

    2010-01-01

    Purpose The toxicity of antiglaucoma medications to ocular surface cells has been evaluated extensively; however, the toxicity to corneal endothelial cells (CECs) remains elusive. Our aim is to evaluate the toxicity of antiglaucoma medications to CECs using an in vitro toxicity assay. Methods Primary cultures of human (H) CECs derived from eye bank specimens were established. Following exposure of HCECs to test solutions for 10, 30, or 60 minutes, or 48 hours, we measured cell viability using a WST-1 assay. Test solutions were diluted in culture media and included 0.5% Timoptol®, preservative-free 0.5% timolol maleate, 1% Trusopt®, preservative-free 1% dorzolamide, Travatan®, Travatan Z®, Xalatan®, and benzalkonium chloride (BAK). To assess cell viability, the value of the test culture well after treatment was expressed as a percentage of that of the control well. Toxicity of each solution was compared using the cell viability score (CVS). Results After exposure to 10-fold dilutions of test solutions for 48 hours, HCEC viabilities were 48.5% for 0.5% Timoptol, 80.9% for preservative-free 0.5% timolol maleate, 47.0% for 1% Trusopt, 71.7% for preservative-free 1% dorzolamide, 55.5% for Travatan, 88.5% for Travatan Z, and 52.5% for Xalatan. Exposure to test solutions diluted 100-fold or more resulted in HCEC viabilities >80%, with the exception of preservative-free 1% dorzolamide, which resulted in a viability of 72.0% at a dilution of 100-fold. Based on CVS, the order of cell viability was Travatan Z ≥ preservative-free timolol maleate = preservative-free dorzolamide > 0.5% Timoptol = 1% Trusopt > Travatan ≥ Xalatan. Assessment of the combined effect of drug and BAK revealed that latanoprost reduced the toxicity of BAK. Conclusion Antiglaucoma eye drops produced HCEC toxicity that appeared to depend on the presence of BAK. Because dilution of the antiglaucoma solutions resulted in markedly lower HCEC toxicity, HCEC damage due to antiglaucoma medication may

  16. Hyaluronic acid concentration-mediated changes in structure and function of porous carriers for corneal endothelial cell sheet delivery.

    Science.gov (United States)

    Lai, Jui-Yang

    2016-02-01

    In this study, the effects of hyaluronic acid (HA) concentrations (0.05-1.25wt.%) on the properties of porous carriers for corneal endothelial tissue engineering were investigated. The pore size and porosity gradually increased with decreasing solid content. However, at relatively low HA concentration (i.e., 0.05wt.%), the material samples contained small interior pores and a dense surface skin layer, probably due to no gas bubble effect on the stirring processing of porous microstructures of freeze-dried polysaccharide hydrogels. The carriers prepared from 0.25wt.% HA solution had the highest freezable water content and oxygen and glucose permeability among the samples evaluated. Results of cell viability assays and quantitative real-time reverse transcription polymerase chain reaction analyses showed that the HA concentration-related alteration of porous microstructure dictates the compatibility of biopolymer carriers with corneal endothelial cell (CEC) cultures. In vivo studies demonstrated that the CEC sheet/HA carrier construct implants are therapeutically efficacious in the reconstruction of endothelial scrape-wounded corneas. It is concluded that the polysaccharide concentration is the major factor for affecting the processing of carriers and their structure and function. Porous hydrogels prepared from 0.25wt.% HA solution are capable of delivering bioengineered CEC sheets to the posterior surface of cornea.

  17. Methods Development for the Isolation and Culture of Primary Corneal Endothelial Cells

    Science.gov (United States)

    2017-02-01

    stromal fibroblasts (keratocytes). Additional challenges are encountered in attempts to expand isolated cells in culture while maintaining CEC morphology...maintain normal cobblestone morphology and favorable growth. However, no results were reported in this study for cells that had been expanded in...and development. Prog Mol Biol Transl Sci 2015; 134: 7-23. 2. Joyce NC. Proliferative capacity of the corneal endothelium. Progress in Retinal and

  18. Micro- and nano-topography to enhance proliferation and sustain functional markers of donor-derived primary human corneal endothelial cells.

    Science.gov (United States)

    Muhammad, Rizwan; Peh, Gary S L; Adnan, Khadijah; Law, Jaslyn B K; Mehta, Jodhbir S; Yim, Evelyn K F

    2015-06-01

    One of the most common indications for corneal transplantation is corneal endothelium dysfunction, which can lead to corneal blindness. Due to a worldwide donor cornea shortage, alternative treatments are needed, but the development of new treatment strategies relies on the successful in vitro culture of primary human corneal endothelial cells (HCECs) because transformed cell lines and animal-derived corneal endothelial cells are not desirable for therapeutic applications. Primary HCECs are non-proliferative in vivo and challenging to expand in vitro while maintaining their characteristic cell morphology and critical markers. Biochemical cues such as growth factors and small molecules have been investigated to enhance the expansion of HCECs with a limited increase in proliferation. In this study, patterned tissue culture polystyrene (TCPS) was shown to significantly enhance the expansion of HCECs. The proliferation of HCECs increased up to 2.9-fold, and the expression amount and localization of cell-cell tight junction protein Zona Occludens-1 (ZO-1) was significantly enhanced when grown on 1 μm TCPS pillars. 250 nm pillars induced an optimal hexagonal morphology of HCEC cells. Furthermore, we demonstrated that the topographical effect on tight-junction expression and cell morphology could be maintained throughout each passage, and was effectively 'remembered' by the cells. Higher amount of tight-junction protein expression was maintained at cell junctions when topographic cues were removed in the successive seeding. This topographic memory suggested topography-exposed/induced cells would maintain the enhanced functional markers, which would be useful in cell-therapy based approaches to enable the in situ endothelial cell monolayer formation upon delivery. The development of patterned TCPS culture platforms could significantly benefit those researching human corneal endothelial cell cultivation for cell therapy, and tissue engineering applications.

  19. Further analysis of assessments of the coefficient of variation of corneal endothelial cell areas from specular microscopic images.

    Science.gov (United States)

    Doughty, Michael J; Aakre, Bente Monica

    2008-09-01

    The aim of this study was to compare two methods of assessments of the coefficient of variation (COV) of endothelial cell area. A single image (Topcon SP-2000P specular microscope) was obtained from the central region of the corneal endothelium of 45 healthy white (Norwegian) individuals, aged from 24 to 43 years and without a history of major eye disease or surgery. The image file was printed to A3-size, the cell-cell boundaries marked manually and the areas of the cells measured with a digitiser pad. The same image file was independently processed by the semi-automated Topcon IMAGEnet system. From either method, the cell area data from 100 contiguous cells approximately in the middle portion of the images were used to calculate the average cell area (AVG), the coefficient of variation (COV) on the cell areas and the endothelial cell density (ECD). Both methods produced similar AVG and ECD values that were not statistically different (p >or= 0.180). The SD values on the cell areas increased in relation to the AVG values (Pearson's r >or= 0.557). The resultant COV values were only marginally higher with the manual method (27.8 versus. 26.3 per cent) but the limits of agreement (LoA) for the COV values were rather large at -4.9 to +7.9 per cent. A semi-automated image analysis system can be used to generate COV data for the corneal endothelium similar to those of a manual method. The limits of agreement between the methods are substantial and this probably reflects the extreme sensitivity of the COV calculation to even a few different cell area values. This poor agreement needs to be considered in any comparative studies.

  20. Corneal endothelial cell loss in post-penetrating keratoplasty patients after cataract surgery: phacoemulsification versus planned extracapsular cataract extraction.

    Science.gov (United States)

    Acar, Banu Torun; Buttanri, Ibrahim Bulent; Sevim, Mehmet Sahin; Acar, Suphi

    2011-08-01

    To compare the changes in endothelial cell density (ECD) in post-penetrating keratoplasty (PKP) patients after cataract extraction with phacoemulsification or planned extracapsular cataract extraction (ECCE). Haydarpasa Numune Education and Research Hospital, Ophthalmology Clinic, Istanbul, Turkey. Clinical trial. Eyes with hard nuclear cataract that had previous PKP were randomly assigned to have phacoemulsification or ECCE. Noncontact specular microscopy was performed preoperatively and 1, 3, and 6 months postoperatively. Twenty-six eyes of 26 patients were enrolled (14 phacoemulsification; 12 ECCE). Six months postoperatively, the mean corneal ECD was statistically significantly lower in the phacoemulsification group (1869.50 cells/mm(2) ± 158.05 [SD]) than in the ECCE group (1996.00 ± 127.96 cells/mm(2)) (P=.024). The mean percentage of endothelial cell loss at 6 months was 20.3% and 12.7%, respectively (P.05). Extracapsular cataract extraction seemed to cause less endothelial cell damage than phacoemulsification in post-PKP patients with hard nuclear cataract. Copyright © 2011 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

  1. Research progress on corneal endothelial cells regeneration%角膜内皮细胞再生研究新进展

    Institute of Scientific and Technical Information of China (English)

    朱梦玉; 邵春益; 傅瑶

    2014-01-01

    Human corneal endothelial cells play a key role in maintaining corneal transparency. Division of the cells cease and arrest in G1 phase when cell-cell contact formed, but they don’t exit the cell cycle. There are many factors involved in corneal endothelial cell proliferation, TGF-β2 inhibits human corneal endothelial cells into the S phase of the cell cycle, whereas EGF, FGF, NGF can promote cell proliferation;ROCK inhibitor Y-27632 can promote adhesion of endothelial cells. Culture of corneal endothelial precursor cells, and inducing pluripotent stem cells into corneal endothelial cell may provide a new method for the treatment of corneal endothelial dysfunction.%人角膜内皮细胞的主要功能是维持角膜透明性,角膜内皮单层发育成熟形成细胞接触后,内皮细胞会停止分裂增殖,但并没有退出细胞周期。角膜内皮细胞的增殖有多种因素的参与和影响,接触抑制和G1期抑制使细胞增殖暂时停止;细胞因子TGF-β2抑制人角膜内皮细胞进入细胞周期S期,而EGF、FGF、NGF则能够促进细胞的增殖;ROCK抑制剂Y-27632能够促进角膜内皮细胞的粘连,有助于内皮细胞的损伤修复。体外培养角膜内皮前体细胞、诱导多潜能干细胞向角膜内皮细胞分化,为今后治疗角膜内皮失代偿提供了新方向。

  2. Comparison of early corneal endothelial cell loss after coaxial phacoemulsification through 1.8 mm microincision and bimanual phacoemulsification through 1.7 mm microincision.

    Science.gov (United States)

    Wilczynski, Michal; Supady, Ewa; Loba, Piotr; Synder, Aleksandra; Palenga-Pydyn, Dorota; Omulecki, Wojciech

    2009-09-01

    To compare corneal endothelial cell loss after coaxial 1.8 mm microincision cataract surgery (MICS) and bimanual 1.7 mm MICS. Department of Ophthalmology, Medical University of Lodz, Lodz, Poland. The study comprised a nonrandomized prospective consecutive series of 51 eyes of 51 patients who had coaxial MICS with implantation of an MI60 foldable intraocular lens (IOL) using a 1.8 mm temporal clear corneal microincision. Fifty eyes of 50 patients who had uneventful bimanual MICS through a 1.7 mm temporal clear corneal incision for a sleeveless phaco tip and a side port for an irrigating chopper with a foldable Acri.Smart 48S foldable IOL implantation served as a reference group. Corneal endothelial cell density, intraoperative phaco power, effective phaco time, and preoperative and postoperative visual acuities were evaluated. The measurements were performed in a semiautomated masked manner. Statistical analysis was done using nonparametric tests (Wilcoxon signed rank test and Mann-Whitney U test). The patients were examined preoperatively and 2 weeks to 1 month postoperatively. The mean follow-up was 22.58 days +/- 5.08 (SD). Postoperatively, the mean corrected distance visual acuity (CDVA)was 0.95 +/- 13 in both groups. There was a significant decrease in endothelial cell density in both groups, 9.46% in Group 1 and 9.27% in Group 2. The between-group difference was not statistically significant (P>.05, Mann-Whitney U test). The visual results were excellent in both groups. Both MICS techniques enabled preservation of corneal endothelial cells equally well and were similar in terms of minor surgical trauma and the influence of surgery on corneal endothelial cell density. Our results support the use of both MICS techniques for cataract surgery.

  3. Research and analysis on corneal endothelial cell morphology and corneal thickness in patients with diabetics%糖尿病患者角膜内皮细胞形态学和角膜厚度研究分析

    Institute of Scientific and Technical Information of China (English)

    徐武平; 魏春惠; 顾榴丽

    2014-01-01

    AIM: To assess the impact of diabetes on corneal endothelial cells through the quantitative analysis of corneal endothelial cell morphology for patients with diabetics. METHODS: The corneal thickness and endothelial cell morphology of 360 eyes of 299 cases were detected using full automatic corneal endothelial cell analyzer. The normal control group included 175 eyes of 148 cases, and there were 185 eyes of 151 cases for the patients with diabetes, 110 eyes of 92 cases for the non-proliferating phase group and 75 eyes of 59 cases for the proliferating phase group. The average density of central corneal endothelial cells, proportion of hexagonal cells, coefficient of variation and corneal thickness were compared among groups, and then the statistical analysis was conducted. RESULTS: Compared with the cornea of the normal group, in the diabetes group, the coefficient of variation of corneal endothelial cells and central corneal thickness increased, while the average density of central corneal endothelial cells and proportion of hexagonal cells decreased, showing a significant difference (P0. 05). CONCLUSION: Compared with the cornea of normal control group, in the diabetes group, the corneal endothelial cells show abnormal morphology, which aggravates with the severity of lesions, especially for the significant changes in the coefficient of variation and the proportion of hexagonal cells. As a result, the corneal resistance to damage in patients with diabetes will decrease.%目的:通过对糖尿病患者进行角膜内皮细胞形态学定量分析,评估糖尿病对角膜内皮细胞的影响。  方法:应用全自动角膜内皮细胞分析仪对299例360眼进行角膜厚度及内皮细胞形态检测。正常对照组148例175眼,糖尿病患者151例185眼,其中非增殖期组患者92例110眼,增殖期组59例75眼。比较各组患者的中央角膜内皮细胞平均密度、六边形细胞比例、变异系数及角膜厚度,并进行统

  4. Optimized human platelet lysate as novel basis for a serum-, xeno- and additive-free corneal endothelial cell and tissue culture.

    Science.gov (United States)

    Thieme, Daniel; Reuland, Lynn; Lindl, Toni; Kruse, Friedrich; Fuchsluger, Thomas

    2017-09-21

    The expansion of donor derived corneal endothelial cells is a promising approach for regenerative therapies in corneal diseases. To achieve the best GMP standard the entire cultivation process should be devoid of non-human components. However, so far there is no suitable xeno-free protocol for clinical applications. We therefore introduce a processed variant of a platelet lysate for the use in corneal cell and tissue culture based on a GMP-grade thrombocyte concentrate. This processed human platelet lysate (phPL), free of any animal components and of anti-coagulants like heparin with a physiological ionic composition, was used to cultivate corneal endothelial cells (EC) in vitro and ex vivo in comparison to standard cultivation with FCS. Human donor corneas were cut in quarters while two quarters of each cornea were incubated with the respective medium supplement. Three fields of view per quarter were taken into account for the analysis. Evaluation of phPL as a medium supplement in cell culture of immortalized EC showed a superior viability compared to fetal calf serum (FCS) control with reduced cell proliferation. Furthermore, the viability during the expansion of primary cells is significantly (3fold+-0.5) increased with phPL compared to FCS standard medium. Quartering donor corneas was traumatic for the endothelium and therefore resulted in increased EC loss. Interestingly, however, cultivation of the quartered pieces for two weeks in 0.1mg/mL pHPL in Biochrome I showed a 21 (+-10) % EC loss compared to 67 (+-12) % EC loss when cultivated in 2% FCS in Biochrome I. The cell culture protocol with pHPL as FCS replacement seems to be superior to the standard FCS protocols with respect to EC survival. It offers a xeno-free and physiological environment for corneal endothelial cells. This alternative cultivation protocol could facilitate the use of EC for human corneal cell therapy. This article is protected by copyright. All rights reserved.

  5. Research progress of cultivation and identification of human corneal endothelial cell in vitro%人角膜内皮细胞的体外培养及其鉴定的研究进展

    Institute of Scientific and Technical Information of China (English)

    贺美宁; 刘二华; 谭钢

    2014-01-01

    Corneal transparence and thickness mostly depend on corneal endothelial cells. The shortage of transplant -grade donor corneal tissues and limited in vitro expansion of human corneal endothelial cells prompted further impetus for the development of tissue-engineered human corneal endothelium reconstructed in vitro. The culture method of human corneal endothelial cell has been widely used. The standard used to evaluate and identify the human corneal endothelial cells cultivated in vitro has not been established. The objective of this article is to summarize the further study on identification and cultivation of human corneal endothelial cell in vitro.%角膜内皮细胞对维持角膜的透明性和厚度起着关键性的作用。人体内角膜内皮细胞有限的增殖能力及角膜供体的短缺,使组织工程人角膜内皮的体外重建受到了关注。目前,人角膜内皮细胞的培养方法已基本成熟。但是体外培养的人角膜内皮细胞的功能评价及鉴定标准却尚未建立。本文就人角膜内皮细胞的体外培养及其鉴定的研究进展进行综述。

  6. Optimization of Cultured Human Corneal Endothelial Cell Sheet Transplantation and Post-Operative Sheet Evaluation in a Rabbit Model.

    Science.gov (United States)

    Yamaguchi, Masahiro; Shima, Nobuyuki; Kimoto, Miwa; Ebihara, Nobuyuki; Murakami, Akira; Yamagami, Satoru

    2016-09-01

    To optimize cultured human corneal endothelial cell (cHCEC) sheet transplantation technique for maintenance of cHCEC viability. cHCEC sheets cultured on a collagen scaffold were covered with or without Viscoat® and exposed to humidified air in the incubator. cHCEC sheets with or without Viscoat® were transplanted into cadaveric porcine eyes by the DSAEK technique with full air tamponade and incubated for various time periods. Then cell viability was determined by using the live/dead assay kit. cHCEC sheets with Viscoat® were transplanted into rabbit eyes and the sheets were histologically evaluated before and 14 days after transplantation. A collagen scaffold and Viscoat® were effective for protecting cHCEC from damage due to air exposure in vitro. All cells died after 18 hours of air exposure in porcine eyes in Viscoat® untreated control. In contrast, Viscoat® treatment sustained full cell viability following 2 hours and could maintain approximately 80% viability after 18 hours. In a rabbit model, transplanted cHCEC sheet with Viscoat® maintained cell density at 2803 ± 229 mm(2) (18% cell loss) and expression of N-cadherin, zonula occludens-1, and actin-filament localized to cell boundary as similar as donor HCEC. Viscoat® can contribute to cHCEC protection from damage caused by exposure to air.

  7. Progenitors for the Corneal Endothelium and Trabecular Meshwork: A Potential Source for Personalized Stem Cell Therapy in Corneal Endothelial Diseases and Glaucoma

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    Wing Yan Yu

    2011-01-01

    Full Text Available Several adult stem cell types have been found in different parts of the eye, including the corneal epithelium, conjunctiva, and retina. In addition to these, there have been accumulating evidence that some stem-like cells reside in the transition area between the peripheral corneal endothelium (CE and the anterior nonfiltering portion of the trabecular meshwork (TM, which is known as the Schwalbe's Ring region. These stem/progenitor cells may supply new cells for the CE and TM. In fact, the CE and TM share certain similarities in terms of their embryonic origin and proliferative capacity in vivo. In this paper, we discuss the putative stem cell source which has the potential for replacement of lost and nonfunctional cells in CE diseases and glaucoma. The future development of personalized stem cell therapies for the CE and TM may reduce the requirement of corneal grafts and surgical treatments in glaucoma.

  8. Treatment with retinoic acid and lens epithelial cell-conditioned medium in vitro directed the differentiation of pluripotent stem cells towards corneal endothelial cell-like cells.

    Science.gov (United States)

    Chen, Ping; Chen, Jun-Zhao; Shao, Chun-Yi; Li, Chuan-Yin; Zhang, Yi-Dan; Lu, Wen-Juan; Fu, Yao; Gu, Ping; Fan, Xianqun

    2015-02-01

    Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have extensive self-renewal capacity and the potential to differentiate into all tissue-specific cell lineages, including corneal endothelial cells (CECs). They are a promising prospect for the future of regenerative medicine. The method of derivation of CECs from ESCs and iPSCs, however, remains to be elucidated. In this study, mouse ESCs and iPSCs were induced to differentiate into CECs using CEC embryonic development events as a guide. All-trans retinoic acid (RA) treatment during the embryoid body (EB) differentiation step was used to promote neural crest (NC) cell differentiation as first step and was followed by a second induction in CEC- or lens epithelial cell (LEC)-conditioned medium (CM) to ultimately generate CEC-like cells. During the corresponding differentiation stages, NC developmental markers and CEC differentiation markers were detected at the protein level using immunocytochemistry (ICC) and at the mRNA level by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). During the first stage, the data indicated that 4 days of treatment with 1 μM RA starting on day 4 of EB formation favored NC cell differentiation and that plating on gelatin-coated plates led to cell migration out of the EBs. The second-stage differentiation results showed that the CM, particularly the LEC-CM, enhanced the yield of polygonal cells with CEC-specific marker expression shown by ICC and RT-qPCR. This study demonstrates that mouse ESCs and iPSCs were induced and expressed CEC differentiation markers when subjected to a two-step inducement process, suggesting that they are a promising resource for corneal endothelium failure replacement therapy in the future.

  9. Evaluation of the Endothelial Cell Density and the Central Corneal Thickness in Pseudoexfoliation Syndrome and Pseudoexfoliation Glaucoma

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    Bożydar T. Tomaszewski

    2014-01-01

    Full Text Available Purpose. Evaluation of central corneal thickness (CCT and endothelial cell density (ECD in patients with senile cataract and coexisting pseudoexfoliation (PEX syndrome with glaucoma (PEXG and without glaucoma using specular microscopy. Participants and Methods. The study included 122 patients (217 eyes. In this group of patients we identified 133 eyes with PEX syndrome (65 with glaucoma, 68 without glaucoma and 84 eyes without PEX syndrome. ECD and CCT were measured in each eye by specular microscopy. Results. ECD in eyes with PEX syndrome without glaucoma (2297 ± 359 cell/mm2 and in eyes with PEXG (2241 ± 363 cell/mm2 was lower than in the control group (2503 ± 262 cell/mm2 (P<0.001. CCT in eyes with PEXG (508.2 ± 32.6 μm was thinner than in eyes with PEX syndrome without glaucoma (529.7 ± 30.3 μm and control group (527.7 ± 29.4 μm (P<0.001. Conclusions. This research shows that in eyes with PEX syndrome, both with and without glaucoma, ECD was statistically significantly lower than in the control group. In patients with PEXG, CCT was statistically significantly thinner than in the PEX syndrome and control group.

  10. Cloning, expression and functional analyses of human platelet-derived growth factor-B chain peptide for wound repair of cat corneal endothelial cells

    Institute of Scientific and Technical Information of China (English)

    LUO Wen-juan; ZHAO Gui-qiu; WANG Chuan-fu; WANG Li-mei; WANG Xiao-ji

    2009-01-01

    Objective: To investigate the biological function of platelet-derived growth factor B (PDGF-B) on the survival and proliferation of cat corneal endothelial cells so as to provide bases for further studies of its role in wound repair and its clinical application.Methods: Total RNA was extracted from the placenta tissues of healthy pregnant women undergoing hysterotokotomy and PDGF eDNA was obtained with re-verse transcription-polymerase chain reaction (RT-PCR). The prokaryotic expression vector pET-PDGF-B was constructed and expressed the recombinant PDGF-B in Escherichia coli (E.coli) BL21 (DE3). After purification and refolding on Ni2+-chelation affinity chromatography (NTA) column, it was used to culture cat corneal endothelial cells. Cell proliferation was tested by modified tertrazolium salt (MTT) and flow cytometer. And the morphologic change and the ultrastructure were ob-served under an inverted phase contrast microscope, a scan-ning electron microscope and a transmission electon microscope, respectively.Results: PDGF-B chain peptide (PDGF-BB) gene was successfully inserted into the prokaryotic expression vector, pET-28a(+). The purified recombined protein pET-PDGF-B showed a single band on sodium dodecyl sulfate polyacry-lamide gel electropheresis (SDS-PAGE) with the molecular weight of about 27 u, which was in agreement with the de-duced value. MTT and flow cytometry showed that PDGF-BB promoted the survival and proliferation of cat corneal en-dothelial cells.Conclusions: The construction of recombinant prokary-otic expression vector pET-PDGF-B and the preparation of PDGF-BB protein provide a foundation for further study of the function of PDGF-BB and producing biological PDGF-BB protein. The expressed PDGF-BB promotes the prolif-eration of cultured cat corneal endothelial cells.

  11. Successful transplantation of in vitro expanded human corneal endothelial precursors to corneal endothelial surface using a nanocomposite sheet

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    Parikumar P

    2011-01-01

    Full Text Available Background: Though the transplantation of in vitro expanded human corneal endothelial precursors in animal models of endothelial damage by injecting into the anterior chamber has been reported, the practical difficulties of accomplishing such procedure in human patients have been a hurdle to clinical translation. Here we report the successful transplantation of in vitro expanded human corneal precursor cells to an animal eye using a transparent Nano-composite sheet and their engraftment.Materials and Methods: Human Corneal endothelial cells (HCEC were isolated from human cadaver eyes with informed consent and expanded in the lab using a sphere forming assay in a novel Thermoreversible Gelation Polymer (TGP for 26 days. HCEC obtained by sphere forming assay were seeded in a novel Nano-composite sheet, which was made of PNIPA-NC gels by in-situ, free-radical polymerization of NIPA monomer in the presence of exfoliated clay (synthetic hectorite “Laponite XLG” uniformly dispersed in aqueous media. After a further seven days in vitro culture of HCEC in the Nano-composite sheet, cells were harvested and transplanted on cadaver-bovine eyes (n=3. The cells were injected between the corneal endothelial layer and the Nano-composite sheet that had been placed prior to the injection in close proximity to the endothelial layer. After three hours, the transplanted Nano-composite sheets were removed from the bovine eyes and subjected to microscopic examination. The corneas were subjected to Histo-pathological studies along with controls. Results: HCEC formed sphere like colonies in TGP which expressed relevant markers as confirmed by RT-PCR. Microscopic studies of the Nanosheets and histopathological studies of the cornea of the Bull’s eye revealed that the HCEC got engrafted to the corneal endothelial layer of the bovine eyes with no remnant cells in the Nanosheet. Conclusion: Transplantation of in vitro expanded donor human corneal endothelial cells

  12. The Study on Improved Cryopreservation Technique of The Ultrastructure of Corneal Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    1994-01-01

    The traditional corneal cryopreservation technique was improved. We carried out an experimental study that rabbit corneas were cryop-reserved by using polyvinylpyrolidone (PVP) as cryoprotective agent and dimethlsulfoxide (DMSO) as the control. The endothelia of cryopreserved corneas were evaluated by scanning and transmission electron microscopy and vital staining. The study shows that PVP is an excellent extracellular cryoprotective agent and has the characteristic of low toxicity or no toxicity to co...

  13. First Identification of a Triple Corneal Dystrophy Association: Keratoconus, Epithelial Basement Membrane Corneal Dystrophy and Fuchs' Endothelial Corneal Dystrophy

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    Cosimo Mazzotta

    2014-09-01

    Full Text Available Purpose: To report the observation of a triple corneal dystrophy association consisting of keratoconus (KC, epithelial basement membrane corneal dystrophy (EBMCD and Fuchs' endothelial corneal dystrophy (FECD. Methods: A 55-year-old male patient was referred to our cornea service for blurred vision and recurrent foreign body sensation. He reported bilateral recurrent corneal erosions with diurnal visual fluctuations. He underwent corneal biomicroscopy, Scheimpflug tomography, in vivo HRT confocal laser scanning microscopy and genetic testing for TGFBI and ZEB1 mutations using direct DNA sequencing. Results: Biomicroscopic examination revealed the presence of subepithelial central and paracentral corneal opacities. The endothelium showed a bilateral flecked appearance, and the posterior corneal curvature suggested a possible concomitant ectatic disorder. Corneal tomography confirmed the presence of a stage II KC in both eyes. In vivo confocal laser scanning microscopy revealed a concomitant bilateral EBMCD with hyperreflective deposits in basal epithelial cells, subbasal Bowman's layer microfolds and ridges with truncated subbasal nerves as pseudodendritic elements. Stromal analysis revealed honeycomb edematous areas, and the endothelium showed a strawberry surface configuration typical of FECD. The genetic analysis resulted negative for TGFBI mutations and positive for a heterozygous mutation in exon 7 of the gene ZEB1. Conclusion: This is the first case reported in the literature in which KC, EBMCD and FECD are present in the same patient and associated with ZEB1 gene mutation. The triple association was previously established by means of morphological analysis of the cornea using corneal Scheimpflug tomography and in vivo HRT II confocal laser scanning microscopy.

  14. [HCO3-]-regulated expression and activity of soluble adenylyl cyclase in corneal endothelial and Calu-3 cells

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    Cui Miao

    2004-04-01

    Full Text Available Abstract Background Bicarbonate activated Soluble Adenylyl Cyclase (sAC is a unique cytoplasmic and nuclear signaling mechanism for the generation of cAMP. HCO3- activates sAC in bovine corneal endothelial cells (BCECs, increasing [cAMP] and stimulating PKA, leading to phosphorylation of the cystic fibrosis transmembrane-conductance regulator (CFTR and increased apical Cl- permeability. Here, we examined whether HCO3- may also regulate the expression of sAC and thereby affect the production of cAMP upon activation by HCO3- and the stimulation of CFTR in BCECs. Results RT-competitive PCR indicated that sAC mRNA expression in BCECs is dependent on [HCO3-] and incubation time in HCO3-. Immunoblots showed that 10 and 40 mM HCO3- increased sAC protein expression by 45% and 87%, respectively, relative to cells cultured in the absence of HCO3-. Furthermore, 40 mM HCO3- up-regulated sAC protein expression in Calu-3 cells by 93%. On the other hand, sAC expression in BCECs and Calu-3 cells was unaffected by changes in bath pH or osmolarity. Interestingly, BCECs pre-treated with10 μM adenosine or 10 μM forskolin, which increase cAMP levels, showed decreased sAC mRNA expression by 20% and 30%, respectively. Intracellular cAMP production by sAC paralleled the time and [HCO3-]-dependent expression of sAC. Bicarbonate-induced apical Cl- permeability increased by 78% (P 3-. However for cells cultured in the absence of HCO3-, apical Cl- permeability increased by only 10.3% (P > 0.05. Conclusion HCO3- not only directly activates sAC, but also up-regulates the expression of sAC. These results suggest that active cellular uptake of HCO3- can contribute to the basal level of cellular cAMP in tissues that express sAC.

  15. Preparation of arginine–glycine–aspartic acid-modified biopolymeric nanoparticles containing epigalloccatechin-3-gallate for targeting vascular endothelial cells to inhibit corneal neovascularization

    Science.gov (United States)

    Chang, Che-Yi; Wang, Ming-Chen; Miyagawa, Takuya; Chen, Zhi-Yu; Lin, Feng-Huei; Chen, Ko-Hua; Liu, Guei-Sheung; Tseng, Ching-Li

    2017-01-01

    Neovascularization (NV) of the cornea can disrupt visual function, causing ocular diseases, including blindness. Therefore, treatment of corneal NV has a high public health impact. Epigalloccatechin-3-gallate (EGCG), presenting antiangiogenesis effects, was chosen as an inhibitor to treat human vascular endothelial cells for corneal NV treatment. An arginine–glycine–aspartic acid (RGD) peptide–hyaluronic acid (HA)-conjugated complex coating on the gelatin/EGCG self-assembly nanoparticles (GEH-RGD NPs) was synthesized for targeting the αvβ3 integrin on human umbilical vein endothelial cells (HUVECs) in this study, and a corneal NV mouse model was used to evaluate the therapeutic effect of this nanomedicine used as eyedrops. HA-RGD conjugation via COOH and amine groups was confirmed by 1H-nuclear magnetic resonance and Fourier-transform infrared spectroscopy. The average diameter of GEH-RGD NPs was 168.87±22.5 nm with positive charge (19.7±2 mV), with an EGCG-loading efficiency up to 95%. Images of GEH-RGD NPs acquired from transmission electron microscopy showed a spherical shape and shell structure of about 200 nm. A slow-release pattern was observed in the nanoformulation at about 30% after 30 hours. Surface plasmon resonance confirmed that GEH-RGD NPs specifically bound to the integrin αvβ3. In vitro cell-viability assay showed that GEH-RGD efficiently inhibited HUVEC proliferation at low EGCG concentrations (20 μg/mL) when compared with EGCG or non-RGD-modified NPs. Furthermore, GEH-RGD NPs significantly inhibited HUVEC migration down to 58%, lasting for 24 hours. In the corneal NV mouse model, fewer and thinner vessels were observed in the alkali-burned cornea after treatment with GEH-RGD NP eyedrops. Overall, this study indicates that GEH-RGD NPs were successfully developed and synthesized as an inhibitor of vascular endothelial cells with specific targeting capacity. Moreover, they can be used in eyedrops to inhibit angiogenesis in corneal NV

  16. Preparation of arginine-glycine-aspartic acid-modified biopolymeric nanoparticles containing epigalloccatechin-3-gallate for targeting vascular endothelial cells to inhibit corneal neovascularization.

    Science.gov (United States)

    Chang, Che-Yi; Wang, Ming-Chen; Miyagawa, Takuya; Chen, Zhi-Yu; Lin, Feng-Huei; Chen, Ko-Hua; Liu, Guei-Sheung; Tseng, Ching-Li

    2017-01-01

    Neovascularization (NV) of the cornea can disrupt visual function, causing ocular diseases, including blindness. Therefore, treatment of corneal NV has a high public health impact. Epigalloccatechin-3-gallate (EGCG), presenting antiangiogenesis effects, was chosen as an inhibitor to treat human vascular endothelial cells for corneal NV treatment. An arginine-glycine-aspartic acid (RGD) peptide-hyaluronic acid (HA)-conjugated complex coating on the gelatin/EGCG self-assembly nanoparticles (GEH-RGD NPs) was synthesized for targeting the αvβ3 integrin on human umbilical vein endothelial cells (HUVECs) in this study, and a corneal NV mouse model was used to evaluate the therapeutic effect of this nanomedicine used as eyedrops. HA-RGD conjugation via COOH and amine groups was confirmed by (1)H-nuclear magnetic resonance and Fourier-transform infrared spectroscopy. The average diameter of GEH-RGD NPs was 168.87±22.5 nm with positive charge (19.7±2 mV), with an EGCG-loading efficiency up to 95%. Images of GEH-RGD NPs acquired from transmission electron microscopy showed a spherical shape and shell structure of about 200 nm. A slow-release pattern was observed in the nanoformulation at about 30% after 30 hours. Surface plasmon resonance confirmed that GEH-RGD NPs specifically bound to the integrin αvβ3. In vitro cell-viability assay showed that GEH-RGD efficiently inhibited HUVEC proliferation at low EGCG concentrations (20 μg/mL) when compared with EGCG or non-RGD-modified NPs. Furthermore, GEH-RGD NPs significantly inhibited HUVEC migration down to 58%, lasting for 24 hours. In the corneal NV mouse model, fewer and thinner vessels were observed in the alkali-burned cornea after treatment with GEH-RGD NP eyedrops. Overall, this study indicates that GEH-RGD NPs were successfully developed and synthesized as an inhibitor of vascular endothelial cells with specific targeting capacity. Moreover, they can be used in eyedrops to inhibit angiogenesis in corneal NV mice.

  17. Preparation of arginine–glycine–aspartic acid-modified biopolymeric nanoparticles containing epigalloccatechin-3-gallate for targeting vascular endothelial cells to inhibit corneal neovascularization

    Directory of Open Access Journals (Sweden)

    Chang CY

    2016-12-01

    Full Text Available Che-Yi Chang,1,2,* Ming-Chen Wang,2,* Takuya Miyagawa,1 Zhi-Yu Chen,1 Feng-Huei Lin,3,4 Ko-Hua Chen,5,6 Guei-Sheung Liu,7 Ching-Li Tseng1 1Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei, 2Department of Biomedical Engineering, Chung Yuan Christian University, Taoyuan, 3Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Zhunan, 4Institute of Biomedical Engineering, National Taiwan University, 5Department of Ophthalmology, Taipei Veterans General Hospital, 6Department of Ophthalmology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan; 7Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, Australia *These authors contributed equally to this work Abstract: Neovascularization (NV of the cornea can disrupt visual function, causing ocular diseases, including blindness. Therefore, treatment of corneal NV has a high public health impact. Epigalloccatechin-3-gallate (EGCG, presenting antiangiogenesis effects, was chosen as an inhibitor to treat human vascular endothelial cells for corneal NV treatment. An arginine–glycine–aspartic acid (RGD peptide–hyaluronic acid (HA-conjugated complex coating on the gelatin/EGCG self-assembly nanoparticles (GEH-RGD NPs was synthesized for targeting the αvβ3 integrin on human umbilical vein endothelial cells (HUVECs in this study, and a corneal NV mouse model was used to evaluate the therapeutic effect of this nanomedicine used as eyedrops. HA-RGD conjugation via COOH and amine groups was confirmed by 1H-nuclear magnetic resonance and Fourier-transform infrared spectroscopy. The average diameter of GEH-RGD NPs was 168.87±22.5 nm with positive charge (19.7±2 mV, with an EGCG-loading efficiency up to 95%. Images of GEH-RGD NPs acquired from transmission electron microscopy showed a

  18. Successful transportation of human corneal endothelial tissues without cool preservation in varying Indian tropical climatic conditions and in vitro cell expansion using a novel polymer

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    Srinivas K Rao

    2014-01-01

    Full Text Available Background: Though the transplantation of human corneal endothelial tissue (CET separated from cadaver cornea is in practice, its transportation has not been reported. We report the successful transportation of CET in varying Indian climatic conditions without cool preservation and the in vitro expansion of Human Corneal Endothelial Precursor Cells (HCEPCs using a novel Thermo-reversible gelation polymer (TGP. Materials and Methods: CET from cadaver corneas (n = 67, unsuitable for transplantation, were used. In phase I, CET was transported in Basal Culture Medium (Group I and TGP (Group II and in Phase II, in TGP cocktail alone, from three hospitals 250-2500 km away, to a central laboratory. The transportation time ranged from 6 h to 72 h and the outdoor temperature between 20°C and 41°C. On arrival, CET were processed, cells were expanded upto 30 days in basal culture medium (Group A and TGP scaffold (Group B. Cell viability and morphology were documented and Reverse transcription polymerase chain reaction (RT-PCR characterization undertaken. Results: In Phase I, TGP yielded more viable cells (0.11 × 10 6 cells than Group I (0.04 × 10 6 cells. In Phase II, the average cell count was 5.44 × 10 4 cells. During expansion, viability of HCEPCs spheres in TGP was maintained for a longer duration. The cells from both the groups tested positive for B-3 tubulin and negative for cytokeratins K3 and K12, thereby proving them to be HCEPCs. Conclusion: TGP preserves the CET during transportation without cool preservation and supports in vitro expansion, with a higher yield of HCEPCs, similar to that reported in clinical studies.

  19. Descemet membrane endothelial keratoplasty with a stromal rim in the treatment of posterior polymorphous corneal dystrophy

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    Pavel Studeny

    2012-01-01

    Full Text Available A 20-year-old patient, diagnosed with posterior polymorphous corneal dystrophy, developed corneal edema for which he underwent Descemet membrane endothelial keratoplasty with a stromal rim (DMEK-S in the right eye. No intra- or postoperative complications were noted. At the last follow-up 2 years and 9 months after the procedure, the best corrected visual acuity was 1.0 and endothelial cell density declined from 3533 cells/mm 2 to 1012 cells/mm 2 . Despite the endothelial cell loss, DMEK-S appears to be a good alternative to other surgical techniques for the treatment of corneal endotheliopathies, and it may be of benefit to young patients.

  20. Simultaneous bilensectomy and endothelial keratoplasty for angle-supported phakic intraocular lens-induced corneal decompensation

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    Vikas Mittal

    2011-01-01

    Full Text Available A 40-year-old lady presented with severe endothelial cell loss in both eyes 14 years after angle-supported phakic intraocular lens (AS PIOL implantation. The left eye had severe corneal edema with bullous keratopathy. The right eye had markedly reduced endothelial cell count (655 cells/mm 2 although the cornea was clear. She underwent simultaneous bilensectomy (AS PIOL explantation and phacoemulsification and Descemet′s stripping and endothelial keratoplasty (DSEK in the left eye. Explanted AS PIOL was identified as ZSAL-4 (Morcher, Stuttgart, Germany model. Corneal edema cleared completely in 2 months with a best corrected visual acuity (-2.25 D sph of 20/60. No intervention was done in the right eye. The present case illustrates that AS PIOL-induced endothelial decompensation can be effectively managed by simultaneous bilensectomy and endothelial keratoplasty.

  1. Endothelial keratoplasty for corneal decompensation leaded by a dexamethasone implant dislocation in anterior chamber

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    Fernanda Pacella

    2016-06-01

    Full Text Available Background: Dexamethasone intravitreal implant (DEX largely showed his safety and efficacy for the treatment of cases of macular edema. Even if uncommon, delivery dislocation in anterior chamber has been described in Literaure as complication of the injection procedure, leading to irreversible endothelial cell loss in the majority of cases. We report a case of a 66-year-old man with pain and vision loss in his left eye. The anamnesis revealed a recent intravitreal injection of DEX implant for a persistent cystoid macular edema related to central retinal vein occlusion. Anterior segment examination showed corneal edema and the rod implant adherent to corneal endothelium. A large peripheral iridectomy was evident with retroillumination and IOL appeared good centered in the bag. The implant was removed but corneal decompensation was irreversible. One month later, an endothelial keratoplasty was successfully performed restoring corneal transparency. DEX intravitreal implant can migrate from vitreous cavity to anterior chamber and lead to irreversible corneal decompensation by mechanical and chemical toxicity on corneal endothelium. Removeal of the implant is necessary to avoid total endothelial decompensation. Despite this, in some cases endothelial keratoplasty had to be performed.

  2. Cytocompatibility of Three Corneal Cell Types with Amniotic Membrane

    Institute of Scientific and Technical Information of China (English)

    CHENJian-su; CHENRui; XUJin-tang; DINGYong; ZHAOSong-bin; LISui-lian

    2004-01-01

    Rabbit limbal corneal epithelial cells, corneal endothelial cells and keratocytes were cultured on amniotic membrane. Phase contrast microscope examination was performed daily. Histological and scan electron microscopic examinations were carried out to observe the growth, arrangement and adhesion of cultivated cells. Results showed that three corneal cell types seeded on amniotic membrane grew well and had normal cell morphology. Cultured cells attached firmly on the surface of amniotic membrane. Corneal epithelial cells showed singular layer or stratification. Cell boundaries were formed and tightly opposed. Corneal endothelial cells showed cobblestone or polygonal morphologic characteristics that appeared uniform in size. The cellular arrangement was compact. Keratocytes elongated and showed triangle or dendritic morphology with many intercellular joints which could form networks. In conclusion, amniotic membrane has good scaffold property, diffusion effect and compatibility with corneal cells. The basement membrane side of amniotic membrane facilitated the growth of corneal epithelial cells and endothelial cells and cell junctions were tightly developed. The spongy layer of amniotic membrane facilitated the growth of keratocytes and intercellular joints were rich. Amniotic membrane is an ideal biomaterial for layering tissue engineered cornea.

  3. Fully automated corneal endothelial morphometry of images captured by clinical specular microscopy

    Science.gov (United States)

    Bucht, Curry; Söderberg, Per; Manneberg, Göran

    2010-02-01

    The corneal endothelium serves as the posterior barrier of the cornea. Factors such as clarity and refractive properties of the cornea are in direct relationship to the quality of the endothelium. The endothelial cell density is considered the most important morphological factor of the corneal endothelium. Pathological conditions and physical trauma may threaten the endothelial cell density to such an extent that the optical property of the cornea and thus clear eyesight is threatened. Diagnosis of the corneal endothelium through morphometry is an important part of several clinical applications. Morphometry of the corneal endothelium is presently carried out by semi automated analysis of pictures captured by a Clinical Specular Microscope (CSM). Because of the occasional need of operator involvement, this process can be tedious, having a negative impact on sampling size. This study was dedicated to the development and use of fully automated analysis of a very large range of images of the corneal endothelium, captured by CSM, using Fourier analysis. Software was developed in the mathematical programming language Matlab. Pictures of the corneal endothelium, captured by CSM, were read into the analysis software. The software automatically performed digital enhancement of the images, normalizing lights and contrasts. The digitally enhanced images of the corneal endothelium were Fourier transformed, using the fast Fourier transform (FFT) and stored as new images. Tools were developed and applied for identification and analysis of relevant characteristics of the Fourier transformed images. The data obtained from each Fourier transformed image was used to calculate the mean cell density of its corresponding corneal endothelium. The calculation was based on well known diffraction theory. Results in form of estimated cell density of the corneal endothelium were obtained, using fully automated analysis software on 292 images captured by CSM. The cell density obtained by the

  4. Corneal stem cells and tissue engineering: Current advancesand future perspectives

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Major advances are currently being made in regenerativemedicine for cornea. Stem cell-based therapiesrepresent a novel strategy that may substituteconventional corneal transplantation, albeit there aremany challenges ahead given the singularities of eachcellular layer of the cornea. This review recapitulatesthe current data on corneal epithelial stem cells,corneal stromal stem cells and corneal endothelialcell progenitors. Corneal limbal autografts containingepithelial stem cells have been transplanted in humansfor more than 20 years with great successful rates,and researchers now focus on ex vivo cultures andother cell lineages to transplant to the ocular surface.A small population of cells in the corneal endotheliumwas recently reported to have self-renewal capacity,although they do not proliferate in vivo . Two mainobstacles have hindered endothelial cell transplantationto date culture protocols and cell delivery methods tothe posterior cornea in vivo . Human corneal stromalstem cells have been identified shortly after therecognition of precursors of endothelial cells. Stromalstem cells may have the potential to provide a directcell-based therapeutic approach when injected tocorneal scars. Furthermore, they exhibit the ability todeposit organized connective tissue in vitro and maybe useful in corneal stroma engineering in the future.Recent advances and future perspectives in the field arediscussed.

  5. Corneal endothelial morphology and central thickness in patients with type II diabetes mellitus

    DEFF Research Database (Denmark)

    Storr-Paulsen, Allan; Singh, Amardeep; Jeppesen, Helene;

    2014-01-01

    PURPOSE: To investigate corneal endothelial cell density and morphology in type II diabetic and non-diabetic patients and to relate potential differences to the glycaemic status. METHODS: A prospective clinical study including 107 patients with type II diabetes and 128 non-diabetic patients. Samp...

  6. 应用羊膜上皮干细胞微环境培养人角膜内皮细胞的研究%Microenvironment of amniotic epithelium cells enhances the proliferation of human corneal endothelial cells

    Institute of Scientific and Technical Information of China (English)

    王忠浩; 陈玮; 宋莉; 沙翔垠; 梁轩伟

    2013-01-01

    Objective To establish an effective method to enhance the proliferation of human corneal endothelial cells (HCECs). Methods The culture conditions of HCEC were optimized by utilizing the totipotent characteristics of human amniotic membrane epithelial stem cells (HAEC) to establish the optimal culture microenvironment of HAEC to promote the proliferation of HCEC. The morphology of HCEC was observed by using phase-contrast microscope and transmission electron microscope. MTT assay and Giemsa staining were performed to detect the proliferation of HCEC. The rate of apoptotic cells was investigated by using Hoechst33342 staining assay. Results Compared to the corneal endothelial cells medium (CEM), the microenvironment containing 20% HAEC-conditioned medium and HAEC-HCEC co-culture microenvironment could promote the proliferation of HCEC and could reduce the apoptosis of HCEC. The cells in HAEC-HCEC microenvironment group could be passaged 4 times without lossing their polygonal appearance. Conclusion The HAEC microenvironment could effectively enhance the proliferation of HCEC, maintain the morphology of HCEC, and inhibit the process of apoptosis of HCEC.%目的:建立一种利用羊膜上皮干细胞(human amniotic membrane epithelial cell,HAEC)微环境培养人角膜内皮细胞(human corneal endothelial cells,HCEC)的方法.方法:制备羊膜上皮干细胞微环境培养HCEC,并探讨诱导HCEC增殖的最佳培养微环境,倒置相差显微镜和透射电镜观察培养过程中细胞的形态学变化,MTT和Giemsa染色观察细胞增殖情况,Hoechst33342检测凋亡细胞比例.结果:在HCEC基本培养液(corneal endothelial cell medium,CEM)的基础上添加20% HAEC上清、HAEC-HCEC的微环境可促进HCEC的增殖,减少凋亡,细胞传代能力显著增强,HAEC-HCEC组传至4代仍保持多角形的内皮细胞形态.结论:羊膜上皮干细胞微环境培养可有效提高HCEE的增殖能力,更好地维持HCEC的形态,并能抑制其凋亡进程.

  7. Cutting and Decellularization of Multiple Corneal Stromal Lamellae for the Bioengineering of Endothelial Grafts.

    Science.gov (United States)

    He, Zhiguo; Forest, Fabien; Bernard, Aurélien; Gauthier, Anne-Sophie; Montard, Romain; Peoc'h, Michel; Jumelle, Clotilde; Courrier, Emilie; Perrache, Chantal; Gain, Philippe; Thuret, Gilles

    2016-12-01

    Engineered corneal endothelial grafts able to provide numerous functional endothelial cells for the restoration of corneal transparency would be a worthwhile way of replacing donor tissue, which is extremely scarce. The grafts are simply constructed: a biocompatible thin and transparent carrier colonized by a monolayer of cultured endothelial cells (ECs). Here we describe a process able to obtain appropriate carriers by recycling human corneas unsuitable for graft in their original state, but liable to provide multiple thin lamellae when cut with a femtosecond laser as used in refractive surgery. We selected a robust method of stromal decellularization. To demonstrate that neither this process nor long-term storage hindered cell adherence, lamellae were endothelialized with an EC line. The constructs achieved up to very high EC density (the main quality criterion for regular donor corneas) while remaining transparent and thin. We verified that they could be inserted in the anterior chamber of a human eye, like a conventional endothelial graft. Human decellularized cornea will likely be directly compatible with the recipient cornea and comply with the requirements of health regulatory authorities. This study demonstrates that thin human corneal lamellae could have high potential as carriers in next-generation therapy for endothelial dysfunctions.

  8. 肾纤维囊为载体培养角膜内皮细胞的实验研究%Experimental studies on kidney fiber capsule as Carriers for Cultivating Corneal endothelial cells

    Institute of Scientific and Technical Information of China (English)

    唐光霞; 陈建苏; 徐锦堂

    2011-01-01

    Objective To investigate the feasibility of kidney fiber capsule as carriers for cultivating corneal endothelial cells and study the characteristics of the cultured cells. Methods Amplified corneal endothelial cells were seeded on culture plates or kidney fiber capsule,and cultured in vitro. Growths of endothelial cells on culture plates and kidney fiber capsule were identified with inverted microscope, immuno-fluorescence, HE staining and scanning electron microscopy. Results The results indicated that corneal endothelial cells attached firmly on the surface of kidney fiber capsule , and grew quickly. Endothelial cells showed polygon or hexagon morphology. Cells could keep the instinct morphology and function after several passage, and cells could be cultured for a long time. Growth and proliferation of cells on culture plate were poor. Conclusion Corneal endothelial cells on kidney fiber capsule grow well and with obvious cell morphology. This study provides a simple and efficient way for the cells cultured in vitro.%目的 探索以脱细胞的肾纤维囊作为载体培养角膜内皮细胞并研究角膜内皮细胞特性.方法 将培养扩增的角膜内皮细胞分别接种到培养板和肾纤维囊上进行体外培养,采用倒置显微镜、免疫荧光、HE染色和扫描电镜的方法进行检测,观察角膜内皮细胞在培养板和肾纤维囊上的生长情况.结果 角膜内皮细胞在肾纤维囊上快速贴壁生长并增殖,细胞形态为多角形或六边形,多次传代后细胞仍维持原有的形态和功能,细胞能长期培养.在培养板上培养的角膜内皮细胞贴壁生长和增殖情况稍差.结论 角膜内皮细胞在脱细胞的肾纤维囊载体上生长良好,细胞形态结构明显.本研究为角膜内皮细胞的体外培养提供了简单和高效的方法.

  9. In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition.

    Science.gov (United States)

    Ho, Wei-Ting; Su, Chien-Chia; Chang, Jung-Shen; Chang, Shu-Wen; Hu, Fung-Rong; Jou, Tzuu-Shuh; Wang, I-Jong

    2016-08-20

    Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of β-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

  10. Corneal endothelial changes after accelerated corneal collagen cross-linking in keratoconus and postLASIK ectasia

    Science.gov (United States)

    Badawi, Amani E

    2016-01-01

    Purpose The purpose of this study was to evaluate the effects of accelerated cross-linking (CXL) on corneal endothelium in keratoconus and postlaser-assisted in situ keratomileusis (LASIK) ectasia. Design This study is a prospective nonrandomized controlled study. Setting This study was conducted in Mansoura Ophthalmic Center (Mansoura University) and Al-Mostakbal Ophthalmic Center, Mansoura, Egypt. Methods In total, 40 eyes with progressive keratoconus and 10 eyes with postLASIK ectasia were subjected to an accelerated CXL (10 mW/cm2 for 9 minutes). Qualitative and quantitative analyses of the corneal endothelial cells were conducted before CXL and 3, 6, and 12 months after CXL by using a specular microscope (Tomy EM-3000). Results There was a significant reduction in endothelial cell count particularly at 3 and 6 months postCXL. In addition, the coefficient of variance was also statistically significantly higher at 3 and 6 months postoperatively than the preCXL value. There was a slight change in the percentage of hexagonal cells. Conclusion The use of accelerated CXL (10 mW/cm2 for 9 minutes) has a transient negative impact on endothelial cell density and/or endothelial morphology. PMID:27757009

  11. Application of Rho Kinase Inhibitors for the Treatment of Corneal Endothelial Diseases

    Directory of Open Access Journals (Sweden)

    Naoki Okumura

    2017-01-01

    Full Text Available ROCK (Rho kinase signaling regulates a wide spectrum of fundamental cellular events and is involved in a variety of pathological conditions. It has therefore attracted research interest as a potential therapeutic target for combating various diseases. We showed that inhibition of ROCK enhances cell proliferation, promotes cell adhesion onto a substrate, and suppresses apoptosis of corneal endothelial cells (CECs. In addition, we reported that a ROCK inhibitor enhances wound healing in the corneal endothelium in animal models and in pilot clinical research. We also demonstrated the usefulness of a ROCK inhibitor as an adjunct drug in tissue engineering therapy as it enhances the engraftment of CECs onto recipient corneas. In 2013, we initiated a clinical trial to test the effectiveness of injection of cultured human CECs into the anterior chamber of patients with corneal endothelial decompensation. This paper reviews the accumulating evidence supporting the potency of ROCK inhibitors in clinical use, both as eye drops and as adjunct drugs in cell-based therapies, for the treatment of corneal endothelial decompensation.

  12. Histology of corneal wound healing after deep lamellar endothelial keratoplasty

    Directory of Open Access Journals (Sweden)

    Chaoran Zhang

    2008-03-01

    Full Text Available Chaoran Zhang1, Jianjiang Xu1, Rongjia Chen21Ophthalmology Department, 2Pathology Department, Eye and Ear Nose Throat Hospital, Fudan University, Shanghai, ChinaAbstract: Deep lamellar endothelial keratoplasty (DLEK has become an alternative procedure for bullous keratopathy. Herein, the histopathological characteristics of corneal wound healing after DLEK were reported. A 71-year-old man suffering from left psuedophakia bullous keratopathy received small incision DLEK. Twenty months later, another penetrating keratoplasty (PK was performed because of the graft failure. The histopathology of the corneal button removed during PK demonstrated the regularity of stromal fiber alignment at the graft interface. Fibrotic repair was limited to the peripheral margins.Keywords: Deep lamellar endothelial keratoplasty, DLEK, bullous keratopathy, penetrating keratoplasty

  13. Espessura corneana central e densidade das células endoteliais corneanas centrais após trabeculectomia com e sem mitomicina C Central corneal thickness and central corneal endothelial cells density after trabeculectomy with and without mitomycin C

    Directory of Open Access Journals (Sweden)

    Rafael Vidal Mérula

    2008-02-01

    Full Text Available OBJETIVO: Comparar a espessura corneana central (ECC, a densidade (DCEC e a área média (ACM das células endoteliais corneanas centrais após trabeculectomia (TREC com e sem mitomicina C (MMC. MÉTODOS: Estudo prospectivo. Foram avaliados 29 olhos divididos em dois grupos: TREC com e sem MMC. Realizaram-se os seguintes exames pré-operatoriamente e três e seis meses após a TREC: paquimetria ultra-sônica e microscopia especular de não-contato. RESULTADOS: As variações da ECC, DCEC e ACM dos olhos submetidos a TREC com MMC foram, respectivamente: - 11,9±25,7 µm, -169,5±145,3 células/mm² e 42,5±33,4 µm² (três meses; - 7,5±28,1 µm, - 220,6±200,2 células/mm² e 31,5±65,3 µm² (seis meses. As variações da ECC, DCEC e ACM dos olhos submetidos a TREC sem MMC foram, respectivamente: - 13,9±16,7 µm, 200,8±155,4 células/mm² e 34,6±52,4 µm² (três meses; - 10,2±15,6 µm, - 277,6±195,7 células/mm² e 34,3±41,0 µm² (seis meses. CONCLUSÃO: TREC realizada com MMC, quando comparada a TREC sem MMC, não acarretou maior redução da DCEC central.Também não houve variação na ECC e na ACM nos dois grupos, após seis meses de seguimento.PURPOSE: To assess comparatively the central corneal thickness (CCT, the central corneal endothelial cells density (CECD and mean area (CECAA, of patients who underwent trabeculectomy (Trab with or without mytomicyn C (MMC. METHODS: Prospective study. Twenty-nine eyes were divided in two groups: Trab with or without MMC. Ultrasonic pachymetry and non-contact specular microscopy were performed pre-operatively and three and six months after Trab. The mean value of the CECD and the low value of the CCT were used. RESULTS: In the group of Trab with MMC, the variations of the CCT, CECD and CECAA values were, respectively: - 11.9±25.7 µm, - 169.5±145.3 cells/mm² and 42.5±33.4 µm² (three months; - 7.5±28.1 µm, - 220.6±200.2 cells/mm² and 31.5±65.3 µm² (six months. In the group of Trab

  14. Effect of basic fibroblast growth factor on corneal endothelial cell damage after cataract surgery%bFGF改善白内障手术引发角膜内皮损伤

    Institute of Scientific and Technical Information of China (English)

    孙丹宇; 包赫; 姜仕先

    2016-01-01

    目的:探究碱性成纤维细胞生长因子(bFGF)对白内障手术引发角膜内皮损伤修复的作用。方法采用超声乳化摘除30只兔子(60眼)的晶体,将其分为2组,随机分为bFGF治疗组和对照组。bFGF治疗组滴用bFGF眼用凝胶,对照组滴用相同体积的生理盐水,分别每天滴用3次。观察记录2组眼睛消肿的时间;在手术后不同时间,分别检测2组切口的愈合面积、角膜厚度、角膜内皮细胞密度,分别取各组前房水,检测各组前房水中NO、IL-1、IL-6和TNF-α的含量。结果 bFGF治疗组的眼睛消肿时间较对照组显著减少(P<0.05);bFGF治疗加速了切口愈合速率、角膜厚度和角膜内皮细胞密度的恢复及前房水内NO、IL-1、IL-6和TNF-α的减少。结论 bFGF可改善白内障手术引起的角膜内皮细胞损伤。%Objective To explore the effect of basic fibroblast growth factor (bFGF)on corneal endothelial cell damage after cataract surgery.Methods Thirty rabbits (60 eyes)whose lens extraction were done by phacoemulsification were divided into 2 groups,namely,bF-GF treatment group and control group.bFGF ophthalmic gel was used to treat the eyes 3 times daily in bFGF treatment group,and the saline of the same volume was used to treat the eyes in control group.The time of corneal edema subsidence was recorded;the healing area of inci-sion,corneal thickness,the density of corneal endothelial cells and the NO,IL-1 ,IL-6 and TNF-αcontents of the aqueous sample in anterior chamber were measured.Results The time of corneal edema subsidence of bFGF treatment group decreased significantly compared with con-trol group;bFGF treatment accelerated the healing rate of incision,the recovery of corneal thickness and the density of corneal endothelial cells and the decrease of the NO,IL-1 IL-6 and TNF-αcontents of the aqueous sample in anterior chamber.Conclusion bFGF treatment can accelerate the recovery of the corneal

  15. Morphometric changes of corneal endothelial cells following intracameral air for micro perforation of the Descemet Membrane during big-bubble deep anterior lamellar keratoplasty

    Directory of Open Access Journals (Sweden)

    Ashbala Khattak

    2016-04-01

    Conclusion: The presence of air inside the anterior chamber for a short term may not cause further endothelial cell loss and can be safely performed to prevent postoperative Descemet Membrane detachment in case of micro perforations.

  16. Pars Plana Vitrectomy and Silicone Oil Injection in Phakic and Pseudophakic Eyes; Corneal Endothelial Changes

    Directory of Open Access Journals (Sweden)

    Fereydoun Farrahi

    2014-01-01

    Full Text Available Purpose: To evaluate the effect of silicone oil (SO on the corneal endothelium in SO filled phakic and pseudophakic vitrectomizied eyes. Methods: This prospective comparative consecutive case-control study evaluated the corneal endothelial characteristics of 64 SO filled vitrectomizied eyes (case group as compared to 46 vitrectomizied eyes without SO injection (control group. Endothelial cell densities (ECD, coefficient of variation (CV, and percentage of hexagonal cells (hexagonality at the corneal center were evaluated preoperatively, 1 month and 6 months after surgery using noncontact specular microscopy and were compared between the two groups. Exclusion criteria were previous vitreoretinal surgery, aphakia, any degree of anterior chamber inflammation, SO bubbles in the anterior chamber and increased intraocular pressure in the postoperative period. Results: Six months after SO injection, mean ECD was 2,438.2±327.6 cell/mm 2 in the case group and 2,462.6±361.7 cell/mm 2 in the control group (P = 0.714 and mean hexagonality was 49.6 ± 6.8 and 54.6 ± 8.9, in the case and control groups, respectively (P = 0.004. Six months after operation, CV in the case group was 39.3 ± 5.6 and that in the control group was 35.7 ± 6.4 (P = 0.003. Conclusion: Although the presence of SO in the vitreous cavity of phakic and pseudophakic eyes causes slight reduction in the number of endothelial cells, however it leads to significant changes in endothelial cell morphology. Thus, removal of SO after reaching the desired tamponade effect is recommended.

  17. Structural, Morphological, and Functional Correlates of Corneal Endothelial Toxicity Following Corneal Exposure to Sulfur Mustard Vapor

    Science.gov (United States)

    2013-10-01

    other provision of law , no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a...of the corneal endothe- lium during the acute SM injury and in resolved versus MGK corneas at 8 weeks. METHODS Ethics Statement and Disclaimers The...Endothelial Permeability Rabbits were euthanized 24 hours after exposure. Five minutes after euthanasia , 20 lL of a 0.1 mg/mL solution of AlexaFluor 488

  18. Corneal Cell Morphology in Keratoconus: A Confocal Microscopic Observation

    Science.gov (United States)

    Ghosh, Somnath; Mutalib, Haliza Abdul; Kaur, Sharanjeet; Ghoshal, Rituparna; Retnasabapathy, Shamala

    2017-01-01

    Purpose To evaluate corneal cell morphology in patients with keratoconus using an in vivo slit scanning confocal microscope. Methods A cross-sectional study was conducted to evaluate the corneal cell morphology of 47 keratoconus patients and 32 healthy eyes without any ocular disease. New keratoconus patients with different disease severities and without any other ocular co-morbidity were recruited from the ophthalmology department of a public hospital in Malaysia from June 2013 to May 2014. Corneal cell morphology was evaluated using an in vivo slit-scanning confocal microscope. Qualitative and quantitative data were analysed using a grading scale and the Nidek Advanced Visual Information System software, respectively. Results The corneal cell morphology of patients with keratoconus was significantly different from that of healthy eyes except in endothelial cell density (P = 0.072). In the keratoconus group, increased level of stromal haze, alterations such as the elongation of keratocyte nuclei and clustering of cells at the anterior stroma, and dark bands in the posterior stroma were observed with increased severity of the disease. The mean anterior and posterior stromal keratocyte densities and cell areas among the different stages of keratoconus were significantly different (P 0.05) among the three stages of keratoconus. Conclusion Confocal microscopy observation showed significant changes in corneal cell morphology in keratoconic cornea from normal healthy cornea. Analysis also showed significant changes in different severities of keratoconus. Understanding the corneal cell morphology changes in keratoconus may help in the long-term monitoring and management of keratoconus. PMID:28894403

  19. Central Corneal Thickness, Corneal Endothelial Characteristics and Intraocular Pressure after Pediatric Cataract Surgery

    Directory of Open Access Journals (Sweden)

    Naveed Nilforushan

    2008-11-01

    Full Text Available

    PURPOSE: To investigate central corneal thickness (CCT, endothelial cell characteristics and intraocular pressure (IOP in eyes with prior pediatric cataract surgery and to compare them with eyes of normal age and sex matched controls. METHODS: Specular microscopy CCT and IOP measurements were performed in 31 eyes of 17 patients with prior congenital cataract extraction and 40 eyes of 20 age and sex matched subjects. The mean of three pachymetric and specular microscopic measurements were recorded. IOP was measured using Goldmann applanation tonometry. RESULTS: Mean CCT was 632±45 µm in eyes with prior pediatric cataract surgery vs 546±33 µm in control eyes (P < 0.001, independent t test and Mann Whitney U-test. Mean IOP was 22.1±3.9 mmHg in eyes with prior pediatric cataract surgery and 14.0±1.6 mmHg in the control group (P < 0.001, independent t-test. There was no significant difference between the two groups in cell count, polymegethism and mean cell area of corneal endothelial cells. CONCLUSIONS: Although the corneas were clinically clear and there was no significant difference in endothelial characteristics in eyes with prior pediatric cataract surgery as compared to normal controls, central corneal thickness in the operated eyes was significantly greater. To differentiate actual glaucoma from artifactual IOP

  20. Long-term changes in corneal endothelial morphology after discontinuation of low gas-permeable contact lens wear

    NARCIS (Netherlands)

    Odenthal, M.T.; Gan, I.M.; Oosting, J.; Kijlstra, A.; Beekhuis, W.H.

    2005-01-01

    Low gas-permeable contact lens wear of polymethyl methacrylate or hydroxyethyl methacrylate material is known to cause morphologic abnormalities in the corneal endothelial cell layer. These lenses were widely prescribed and successfully worn until their use was actively discouraged in the late 1980s

  1. Femtosecond laser cutting of multiple thin corneal stromal lamellae for endothelial bioengineering.

    Science.gov (United States)

    Bernard, Aurélien; He, Zhiguo; Forest, Fabien; Gauthier, Anne-Sophie; Peocʼh, Michel; Dumollard, Jean-Marc; Acquart, Sophie; Montard, Romain; Delbosc, Bernard; Gain, Philippe; Thuret, Gilles

    2015-02-01

    To assess the feasibility of cutting multiple thin stromal lamellae in human donor corneas using a commercial femtosecond laser (FSL) to provide cell carriers for future endothelial graft bioengineering. Eight edematous organ-cultured corneas not suitable for grafting for endothelial reasons were mounted on a Ziemer anterior chamber and cut with a Z6 FSL with 6 successive parallel cuts, from depth to surface. Target thickness of each lamella ranged from 100 to 150 μm depending on initial corneal thickness. Thickness was measured using anterior segment optical coherence tomography before and after cutting on mounted corneas, and on each stromal lamella after detachment. Scanning electron microscopy observation was performed on 4 lamellae and histological cross sections on 1 cornea before detachment. A median of 5 (minimum 3, maximum 7) lamellae was obtained per cornea. All lamellae still attached were the most posterior ones, suggesting that FSL was less efficient because of light scattering by edematous stroma. Cut precision and postdetachment swelling were correlated with anterior-posterior position within the cornea. Median lamella thickness was 127 μm (56-222 μm) before detachment and 196 μm (80-304 μm) after detachment. Surface state was consistent with previously reported FSL lamellar cuts during Descemet stripping automated endothelial keratoplasty. Up to 7 thin lamellae can be cut in stored corneas with an FSL. This method, once optimized primarily by using deswelled, more transparent corneas, could prove effective for recycling unsuitable donor corneas in corneal bioengineering processes.

  2. Cataract surgery in a patient with severe chronic iritis and corneal endothelial damage.

    Science.gov (United States)

    Yasukawa, T; Suga, K; Yokoo, N; Asada, S

    1998-07-01

    We report a patient with broad anterior synechias and corneal endothelial damage. The patient had chronic iritis and cataracts secondary to chronic iritis in both eyes. Because the right eye had broad anterior synechias and severe corneal endothelial damage, extracapsular cataract extraction and intraocular lens implantation were performed through the basal iris. Good postoperative visual acuity was obtained. The cornea showed little trauma from the surgery and remained clear 36 months postoperatively.

  3. [Endothelial cell adhesion molecules].

    Science.gov (United States)

    Ivanov, A N; Norkin, I A; Puchin'ian, D M; Shirokov, V Iu; Zhdanova, O Iu

    2014-01-01

    The review presents current data concerning the functional role of endothelial cell adhesion molecules belonging to different structural families: integrins, selectins, cadherins, and the immunoglobulin super-family. In this manuscript the regulatory mechanisms and factors of adhesion molecules expression and distribution on the surface of endothelial cells are discussed. The data presented reveal the importance of adhesion molecules in the regulation of structural and functional state of endothelial cells in normal conditions and in pathology. Particular attention is paid to the importance of these molecules in the processes of physiological and pathological angiogenesis, regulation of permeability of the endothelial barrier and cell transmigration.

  4. Long-lasting corneal endothelial graft rejection successfully reversed after dexamethasone intravitreal implant

    Directory of Open Access Journals (Sweden)

    Giannaccare G

    2016-07-01

    Full Text Available Giuseppe Giannaccare, Michela Fresina, Alberto Pazzaglia, Piera Versura Ophthalmology Unit, Department of Experimental, Diagnostic and Specialty Medicine (DIMES, Alma Mater Studiorum University of Bologna, Sant’Orsola‑Malpighi Teaching Hospital, Bologna, Italy Abstract: Graft rejection is the most significant complication corneal transplantation and the leading indication for overall corneal transplantation. Corticosteroid therapy represents the mainstay of graft rejection treatment; however, the optimal route of administration of corticosteroid remains uncertain. We report herein for the first time the multimodal imaging of a case of long-lasting corneal endothelial graft rejection successfully reversed 3 months after dexamethasone intravitreal implant. A 29-year-old Asian female presented with a long-lasting corneal endothelial graft rejection in her left phakic eye. She underwent penetrating keratoplasty for advanced keratoconus 24 months before presentation. Hourly dexamethasone eyedrops, daily intravenous methylprednisolone, and one parabulbar injection of methylprednisolone acetate were administered during the 5 days of hospitalization. However, the clinical picture remained approximately unchanged despite therapy. By mutual agreement, we opted for the off-label injection of dexamethasone 0.7 mg intravitreal implant in order to provide therapeutic concentrations of steroid for a period of ~6 months. No other concomitant therapies were prescribed to the patient. Visual acuity measurement, slit lamp biomicroscopy, anterior segment photography, confocal microscopy, anterior segment optical coherence tomography, laser cell flare meter, intraocular pressure measurement, and ophthalmoscopy were performed monthly for the first postoperative 6 months. Three months after injection, both clinical and subclinical signs of rejection disappeared with a full recovery of visual acuity to 20/30 as before the episode. Currently, at the 12-month

  5. Regenerative capacity and influential factors of corneal endothelial cells%角膜内皮细胞的再生特性及影响因素

    Institute of Scientific and Technical Information of China (English)

    陈根云; 陈桂强

    2008-01-01

    学术背景:原发性和继发性角膜内皮失代偿是影响角膜移植、白内障手术、青光眼手术等眼科复明后成功率的主要因素之一,其发生的病理生理基础在于成年人角膜内皮细胞失去了有丝分裂能力,损伤后无法再生.目的:介绍角膜内皮细胞再生特性研究现状和影响因素,为角膜病的防治提供新的思路.检索策略:应用计算机检索PubMed数据库1973-01/2007-12的有关文献,检索词为"corneal endothelial cells,regeneration,proliferation,gene transfer",限定文章语言种类为English.同时应用计算机检索中国期刊全文数据库1973-01/2007-12的有关文献,检索词为"角膜内皮细胞,再生,增殖,基因转染",限定文章语言种类为中文.共检索到246篇相关文献,其中中文文献185篇,英文文献61篇.根据文题和研究日的对文献进行筛选.纳入标准:①选取针对性强,相关度高的文献.②对同一领域的文献选择近期发表或权威杂志的文献;排除重复研究和知识较为陈旧的文章.最后32篇论文被选用.文献评价:文献的来源主要是和角膜内皮细胞再生特性、增殖或有丝分裂能力相关的研究,而且是已经在国内、外正规医学期刊公开发表的论文.所选用的32篇文章中6篇为综述,其余26篇均为临床与实验研究.资料综合:①不同种属动物角膜内皮细胞有丝分裂能力不同,而人类角膜内皮细胞在再生能力方面存在年龄差别、在体和离体差别、周边区和中央区差别等特点.②成年人角膜内皮细胞失去有丝分裂能力主要与细胞接触抑制、转化生长因子β2以及细胞因子p27kip1等有密切的关系.③基因转染、生长因子以及细胞外基质等因素能够影响角膜内皮细胞再生特性,是角膜病防治的新方向.结论:通过各种措施调控角膜内皮细胞的生长周期,可望恢复成人角膜内皮细胞的再生能力,是提高角膜移植成功率的新方向.

  6. 小鼠胚胎干细胞条件培养液培养的人角膜内皮细胞在脱细胞猪角膜基质上单层细胞片的构建%Formation of cell sheet on acellular porcine corneal stroma with human corneal endothelial cells cocultured by mouse embryonic stem cell conditioned medium

    Institute of Scientific and Technical Information of China (English)

    鹿晓燕; 王智崇

    2016-01-01

    Background Corneal transplantation faces a great challenge because of the shortage of corneal donors and difficulty of human corneal endothelial cells (HCECs) regeneration in vitro.So the study on tissue engineering cornea is still a main topic.Previous research showed that mouse embryonic stem cell conditioned medium (ESC-CM) improved the proliferative capacity of HCECs in vitro,and acellular porcine corneal stroma (APCS) was a good saffold material.However,whether HECEs cultured by mouse ESC-CM can form cell sheet in vitro were rarely studied.Objective This study was to investigate the potential that HCECs cultured by mouse ESC-CM form a monolayer cell sheet.Methods The supernatant of ESC-CM was collected after mouse ES-E14 cells were cultured,and the cultured medium was centrifuged and mixed with 75% human corneal endothelium medium (CEM)at a proportion of 1 ∶ 3 to prepare the 25% ESC-CM system.Primary cultures of HCECs were established from explants of corneal limbal with Descemet's membrane,and the cells were identified by using reverse-transcription PCR to determine the expressions of collagen Ⅷ (Col Ⅷ) mRNA and neuron-specific enolase (NSE) mRNA in the cells.APCS was prepared by decellularization with phospholipase A2 and bicarbonate solution,and the second generation of HCECs were inoculated on the sterilized APCS at a 800/mm2 density.The morphology of the cells was observed by hematoxylin-eosin staining under the phase-contrast microscope.The expressions of zona occludens protein-1 (ZO-1)and Na+-K+-ATPase in the cell sheet were detected by immunofluorescence staining.Results The second generation of HCECs cultured with 25% ESC-CM in vitro showed the hexagon in shape with positive expressions for Col Ⅷ mRNA and NSE mRNA.Decellularization APCS was transparent,and no corneal cells were seen,the structures of corneal collagenous fibres were regular.HCECs attached closely to APCS and formed monolayer sheet 7 days after culture on the APCS with the

  7. 恒河猴和树鼩角膜内皮细胞的比较分析%Comparison of the corneal endothelial cells of rhesus monkeys and tree shrews

    Institute of Scientific and Technical Information of China (English)

    吴敏; 李娜; 孙晓梅; 胡竹林

    2016-01-01

    的合适实验动物。%Objective To analyze and compare the characteristics and differences of corneal endothelial cells of rhesus monkey and tree shrew eyes.Methods Corneal endothelial cells of 6 healthy rhesus monkeys (12 eyes) and 20 healthy tree shrews (40 eyes) were measured using a non-contact SP3000P specular microscope.Eight parameters were de-termined and compared with relevant parameters of human eyes reported in the literature, including minimum cell area (Smin), maximum cell area (Smax), average cell area (Savg), standard deviation of cell area (SD), coefficient of variabili-ty ( CV) , cell density ( CD) , hexagonality percentage ( HG%) and central corneal thickness ( CCT) .Results The ima-ging and measurement of all parameters could be completed in a short time both in rhesus monkeys and tree shrews.The time spent in the two kinds of animals was not significantly different.The CCT was ( 449.2 ±12.8 ) μm and ( 262.4 ± 24.6) μm, Smin was (120.4 ±26.3) S/μm2 and (153.2 ±42.9) S/μm2 , Smax was (705.0 ±130.8) S/μm2 and (468.7 ±109.3) S/μm2 , Savg was (351.1 ±26.1) and (295.4 ±18.9) S/μm2 , SSD was (113.1 ±27.4) and (75.9 ±27.3) S/μm2, CV was (31.9 ±6.0) and (25.3 ±8.3), CD was (2874.2 ±203.8) p/cell· mm-2 and (3399.3 ±224.7) p/cell· mm-2 , and the HG% was (58.6 ±9.1) and (94.0 ±9.7) in the rhesus monkeys andt tree shrews, respectively. The differences of all the above parameters between rhesus monkeys and tree shrews were statistically significant ( P<0.05 for all) .The cornea of tree shrews was significantly thinner than that of rhesus monkeys.The area and coefficient of varia-bility of tree shrews were smaller to those of rhesus monkeys, while the cell density and hexagonality percentage were higher than those of rhesus monkeys.Compared with human eyes, the CCT, CV and HG%in rhesus monkeys were highly simi-lar, while the CD was lower than that of human eyes.The CCT in tree shrew was only 60%of the rhesus monkey eyes and 50%of human eyes, while the CD and Savg

  8. The preliminary analysis of corneal endothelial cell of Chinese Air Force pilot%空军飞行员角膜内皮细胞的初步分析

    Institute of Scientific and Technical Information of China (English)

    刘兵; 茹海霞; 马海燕; 赵蓉; 邵德望

    2008-01-01

    目的 探讨我国空军飞行员角膜内皮细胞密度和形态特征,初步了解细胞密度和形态学的生理学参数.方法 应用角膜内皮细胞显微镜及仪器自带的角膜内皮细胞分析系统,对男性56例(112眼)现役飞行员角膜内皮细胞进行了观察,同时选取30例(60眼)与其年龄和裸眼视力具有可比性的健康男性作为对照组,两组进行分析和比较.结果 数据显示空军飞行员平均角膜内皮细胞密度为2797.0±330.3个/mm2,显著低于对照组的2917.57±318.04个/mm2(t=2.286,P=0.024).平均六角形细胞构成比,飞行员组(56.46±7.19)显著低于对照组(60.33±7.68),差异有显著性意义(t=3.253,P=0.001).飞行员组和对照组随着年龄的增加,六角形细胞构成比均逐渐减少(r=-0.395、-0.268,P=0.000、0.038).通过偏相关系数分析,在年龄因素控制条件下,飞行员飞行时间的延长伴随着细胞平均面积的增大(r=0.244,P=0.01).结论 对飞行员角膜内皮细胞形态学参数的测定和初步数据分析将有助于进一步了解高速飞行情况下眼角膜内皮的变化.%Objegtive To preliminarily study the endothelial density baseline and the morphological characteristics of the corneal endothelial cell of Chinese Air Force pilot. Metheds Density and morphology of corneal endothelial cells were observed by Topcon-2000 noncontact specular microscope and analyzed by Imagenet 2000 software(version 2.53)on 56 pilots(112 eyes)While other 30 healthy men(60 eyes)who were with the comparable age and uncorrected visual acuity were compared and analyzed as the control group. Results The data showed that the average corneal endothelial cell density was 2797.0±330.3/mm2 in pilot group which was significantly lowet than 2917.57±318.04/mm2 in control group(t=2.286,P=0.024).The mean constituent ratio of hexagonal cell was 56.46±7.19 in pilot group compared with 60.33±7.68 in control group(t=8.253,P=O.001)and it was gradually reduced with the age

  9. 人热敏瞬时受体通道1基因转染对兔角膜内皮细胞的影响%Effects of human thermal transient receptor channel 1 gene transfection on cultured rabbit corneal endothelial cells in vitro

    Institute of Scientific and Technical Information of China (English)

    王莉; 杜兆江; 李鹏

    2015-01-01

    ?AIM:To explore the effects of human thermal transient receptor channel 1 gene transfection on corneal endothelial cell of rabbits. ?METHODS:Research group were dealt for thermal transient receptor channel 1 gene mediated by liposome transfection to rabbit corneal endothelial cells. MTT method was used to observe its influence on cell proliferation. Immunohistochemical staining and computer image analysis system were used to test the effects for proliferation cell nucleus antigen ( PCNA ) expression. ?RESULTS:Proliferation of corneal endothelial cell of rabbit was promoted after thermal transient receptor channel 1 gene transfected and the difference between experiment group and control group (t=3.01,P=0.013). The expression of PCNA promoted after thermal transient receptor channel 1 gene transfected (t=3.21,P=0.007) compared with control group. ? CONCLUSION: The expression of PCNA in rabbit corneal endothelial cells can promote the proliferation of corneal endothelial cells of rabbits.%目的:探讨人热敏瞬时受体通道1基因转染对培养的兔角膜内皮细胞增殖能力的影响。  方法:研究组为人热敏瞬时受体通道1基因通过脂质体介导的方法转染到体外培养的兔角膜内皮细胞中,采用MTT方法观察对细胞增殖的影响,免疫组织化学染色法和计算机图像分析系统检测对细胞增殖细胞核抗原( proliferation cell nucleus antigen,PCNA)表达的影响。  结果:热敏瞬时受体通道1基因转染后内皮细胞增殖增加,实验组与对照组比较差异有统计学意义(t=3.01,P=0.013);实验组细胞PCNA表达明显增加,与对照组比较差异有统计学意义( t=3.21,P=0.007)。  结论:人热敏瞬时受体通道1基因转染可以促进兔角膜内皮细胞增殖。

  10. Confocal microscopy and electrophysiological study of single patient corneal endothelium cell cultures

    Science.gov (United States)

    Tatini, Francesca; Rossi, Francesca; Coppi, Elisabetta; Magni, Giada; Fusco, Irene; Menabuoni, Luca; Pedata, Felicita; Pugliese, Anna Maria; Pini, Roberto

    2016-04-01

    The characterization of the ion channels in corneal endothelial cells and the elucidation of their involvement in corneal pathologies would lead to the identification of new molecular target for pharmacological treatments and to the clarification of corneal physiology. The corneal endothelium is an amitotic cell monolayer with a major role in preserving corneal transparency and in regulating the water and solute flux across the posterior surface of the cornea. Although endothelial cells are non-excitable, they express a range of ion channels, such as voltage-dependent Na+ channels and K+ channels, L-type Ca2 channels and many others. Interestingly, purinergic receptors have been linked to a variety of conditions within the eye but their presence in the endothelium and their role in its pathophysiology is still uncertain. In this study, we were able to extract endothelial cells from single human corneas, thus obtaining primary cultures that represent the peculiarity of each donor. Corneas were from tissues not suitable for transplant in patients. We characterized the endothelial cells by confocal microscopy, both within the intact cornea and in the primary endothelial cells cultures. We also studied the functional role of the purinergic system (adenosine, ATP and their receptors) by means of electrophysiological recordings. The experiments were performed by patch clamp recordings and confocal time-lapse microscopy and our results indicate that the application of purinergic compounds modulates the amplitude of outward currents in the isolated endothelial cells. These findings may lead to the proposal of new therapies for endothelium-related corneal diseases.

  11. 长期配戴角膜塑形镜对角膜内皮细胞的影响观察%Observation of the Influence to corneal endothelial cell from long- term wearing orthokeratology Lenses

    Institute of Scientific and Technical Information of China (English)

    褚春漫

    2011-01-01

    目的:评价近视眼患者长期配戴角膜塑形镜后不同时期角膜内皮细胞密度和形态的情况。方法:用非接触型角膜内皮显微镜对22例(44眼)配戴角膜塑形镜者,在戴镜前、戴镜后1年及2年作角膜内皮细胞照相观察。结果:戴镜前、戴镜后1年及2年角膜内皮细胞密度分别为(3228±168)个/mm2、(3192±172)个/mm2及(3178±181)个/mm2(p〈0.05),戴镜后角膜内皮细胞形态无明显改变。结论:轻、中度近视患者,利用高透氧材料的角膜塑形镜进行科学的角膜塑形治疗,不会对角膜内皮细胞密度和形态带来影响,长期配戴用于控制近视发展基本是安全的。%Objective: To evaluate the comeal endothelial cell density and morphological changes by periods after longterm wearing Orthokeratology Lenses. Method:22 patients (44 eyes) wore orthokeratology lenses were graphically recorded and observed by Non - contact Specular Microscope at the time of before wear, 1 year after wear and 2 years after wear. Results:The coroeal endotheliaf cell density at each time point were (3228 ± 168) cells/mm2 (before wear), ( 3192 ±172 ) cells/mm2 ( 1 year after wear ) and ( 3178 ± 181 ) cells / mm2 ( 2 years after wear } ( P 〉 0. 05 ). Conclusion: For slight and medium myopia patients, using orthokeratology lenses which made of Highly Oxygen - permeable material for comea moulding have no influence to corneal endothelial cell density and morphology, o rthokeratology lenses wearing is a safe way for myopia correction.

  12. Corneal reconstruction by stem cells and bioengineering

    Directory of Open Access Journals (Sweden)

    Arjamaa O

    2012-09-01

    Full Text Available Olli ArjamaaDepartment of Biology, University of Turku, Turku, FinlandAbstract: Almost 300 million people are visually impaired worldwide due to various eye diseases such as cataracts, glaucoma, age-related macular degeneration, diabetic retinopathy, and corneal diseases. Notably, ten million people are blind because of severe ocular surface diseases and the majority of cases occur in developing countries. Blinding ocular surface diseases have, however, become treatable by grafting of surface layers, or by full-thickness transplantation of the cornea. As the demand for human corneal tissue for surface reconstruction and transplantation far exceeds the supply, methods are being developed to supplement tissue donation. Xenotransplantation of the cornea or cells from genetically modified pigs may become one of the solutions. Transplantation of limbal stem cells within tissue biopsies, to restore the transparency of the cornea is another remarkable method, which has shown its potential in several clinical studies. The combination of stem cell technology and engineering of biocompatible tissue equivalent, still at preclinical stage, has shown us how synthetic corneal tissue is able to guide cultured corneal stromal stem cells of human origin, to become native-like stroma, the most important layer of the cornea. These findings give hope for a large-quantity production of biomaterial for corneal reconstruction. As such, clinical ophthalmologists should become more familiar with the methods of laboratory science.Keywords: eye, grafting, keratoplasty, xenotransplantation, cell reservoir, biocompatible tissue equivalent

  13. Trinucleotide Repeat Expansion in the Transcription Factor 4 (TCF4) Gene Leads to Widespread mRNA Splicing Changes in Fuchs' Endothelial Corneal Dystrophy

    Science.gov (United States)

    Wieben, Eric D.; Aleff, Ross A.; Tang, Xiaojia; Butz, Malinda L.; Kalari, Krishna R.; Highsmith, Edward W.; Jen, Jin; Vasmatzis, George; Patel, Sanjay V.; Maguire, Leo J.; Baratz, Keith H.; Fautsch, Michael P.

    2017-01-01

    Purpose To identify RNA missplicing events in human corneal endothelial tissue isolated from Fuchs' endothelial corneal dystrophy (FECD). Methods Total RNA was isolated and sequenced from corneal endothelial tissue obtained during keratoplasty from 12 patients with FECD and 4 patients undergoing keratoplasty or enucleation for other indications. The length of the trinucleotide repeat (TNR) CTG in the transcription factor 4 (TCF4) gene was determined using leukocyte-derived DNA analyzed by a combination of Southern blotting and Genescan analysis. Commercial statistical software was used to quantify expression of alternatively spliced genes. Validation of selected alternative splicing events was performed by using RT-PCR. Gene sets identified were analyzed for overrepresentation using Web-based analysis system. Results Corneal endothelial tissue from FECD patients containing a CTG TNR expansion sequence in the TCF4 gene revealed widespread changes in mRNA splicing, including a novel splicing event involving FGFR2. Differential splicing of NUMA1, PPFIBP1, MBNL1, and MBNL2 transcripts were identified in all FECD samples containing a TNR expansion. The differentially spliced genes were enriched for products that localize to the cell cortex and bind cytoskeletal and cell adhesion proteins. Conclusions Corneal endothelium from FECD patients harbors a unique signature of mis-splicing events due to CTG TNR expansion in the TCF4 gene, consistent with the hypothesis that RNA toxicity contributes to the pathogenesis of FECD. Changes to the endothelial barrier function, a known event in the development of FECD, was identified as a key biological process influenced by the missplicing events. PMID:28118661

  14. Mecanotransduction and Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    S.MULLER; JF.; STOLTZ2

    2005-01-01

    1 IntroductionAtherosclerosis preferentially occurs in areas of complex blood flow where there are disturbed flow and low fluid shear stress, whereas laminar blood flow and high shear stress are atheroprotective~([1]). Reports of others and our studies suggest a steady laminar flow decreases some molecules and genes expression of vascular endothelial cells (EC) that may promote atherosclerosis, as well as it can differentially regulate production of many vasoactive factors at the level of gene expression an...

  15. Putative epidermal stem cell convert into corneal epithelium-like cell under corneal tissue in vitro

    Institute of Scientific and Technical Information of China (English)

    GAO Nan; CUI GuangHui; WANG ZhiChong; HUANG Bing; GE Jian; LU Rong; ZHANG KeFei; FAN ZhiGang; LU Li; PENG Zhan

    2007-01-01

    Rhesus putative epidermal stem cells are being investigated for their potential use in regenerative corneal epithelium-like cells, which may provide a practical source of autologous seed cells for the construction of bioengineered corneas. The goal of this study was to investigate the potential of epidermal stem cells for trans-differentiation into corneal epithelium-like cells. Rhesus putative epidermal stem cells were isolated by type IV collagen attachment method. Flow cytometry analysis, immunohistology and RT-PCR were conducted to identify the expression of specific markers (β1, α6 integrin, K15, K1/K10, K3/K12 and CD71) on the isolated rapid attaching cells. The isolated cells were cocultured with human corneal limbal stroma and corneal epithelial cells. After coculture, the expression of the same specific markers was evaluated in order to identify expression difference caused by the coculture conditions. K3/K12 expression was analyzed in coculture cells on day 2, 4, 6, 8 and 10. Putative epidermal stem cells in conditioned culture media were used as control. Putative epidermal stem cells were predominant in rapid attaching cells by type IV collagen attachment isolation. Before being cocultured, the rhesus putative epidermal stem cells expressed K15, α6 and β1 integrin, but no CD71, K1/K10 and K3/K12. After coculture, these cells expressed K3/K12 (a marker of corneal epithelial cells), K15 and β 1 integrin, but no K1/K10. Cells being not coculture converted into terminally differentiated cells expressing K1/K10. These results indicate that rhesus putative epidermal stem cells can trans-differentiate into corneal epithelium-like cells and, therefore, may have potential therapeutic application as autologous seed cells for the construction of bioengineered corneas.

  16. Putative epidermal stem cell convert into corneal epithelium-like cell under corneal tissue in vitro

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Rhesus putative epidermal stem cells are being investigated for their potential use in regenerative corneal epithelium-like cells, which may provide a practical source of autologous seed cells for the construction of bioengineered corneas. The goal of this study was to investigate the potential of epi-dermal stem cells for trans-differentiation into corneal epithelium-like cells. Rhesus putative epidermal stem cells were isolated by type IV collagen attachment method. Flow cytometry analysis, immuno-histology and RT-PCR were conducted to identify the expression of specific markers (β1, α6 integrin, K15, K1/K10, K3/K12 and CD71) on the isolated rapid attaching cells. The isolated cells were cocultured with human corneal limbal stroma and corneal epithelial cells. After coculture, the expression of the same specific markers was evaluated in order to identify expression difference caused by the coculture conditions. K3/K12 expression was analyzed in coculture cells on day 2, 4, 6, 8 and 10. Putative epi-dermal stem cells in conditioned culture media were used as control. Putative epidermal stem cells were predominant in rapid attaching cells by type IV collagen attachment isolation. Before being co-cultured, the rhesus putative epidermal stem cells expressed K15, α6 and β1 integrin, but no CD71, K1/K10 and K3/K12. After coculture, these cells expressed K3/K12 (a marker of corneal epithelial cells), K15 and β 1 integrin, but no K1/K10. Cells being not coculture converted into terminally differentiated cells expressing K1/K10. These results indicate that rhesus putative epidermal stem cells can trans-differentiate into corneal epithelium-like cells and, therefore, may have potential therapeutic application as autologous seed cells for the construction of bioengineered corneas.

  17. Fuchs endothelial corneal dystrophy: clinical characteristics of surgical and nonsurgical patients

    Directory of Open Access Journals (Sweden)

    Goldberg RA

    2014-09-01

    Full Text Available Roger A Goldberg,1,2 Sabri Raza,1 Eric Walford,1 William J Feuer,1 Jeffrey L Goldberg1,3 1Bascom Palmer Eye Institute, University of Miami, Miami, FL, USA; 2Tufts-New England Eye Center/Ophthalmic Consultants of Boston, Boston, MA, USA; 3Shiley Eye Center, University of California San Diego, San Diego, CA, USA Purpose: To review the patient and clinical characteristics of patients with Fuchs endothelial corneal dystrophy (FECD. Methods: Review of records for every patient who presented to the Bascom Palmer Eye Institute between 2003 and 2009 whose visit was coded for endothelial corneal dystrophy (International Classification of Diseases, Ninth Revision [ICD9] 371.57, bullous keratopathy (ICD9 371.23, or who underwent a corneal surgery with or without cataract extraction. Demographic, clinical, and ancillary testing data were collected from the time of presentation, diagnosis, and follow-up, and the use, timing, and type of surgical interventions was documented, with 6-month and final visual acuities recorded. Results: A total of 2,370 charts were included in this study, of which 966 patients had a diagnosis of FECD. Of these, 197 patients (21% received a corneal transplantation procedure. The surgery most often performed was penetrating keratoplasty with or without cataract extraction (66%, followed by endothelial keratoplasty with or without cataract extraction (34%. The risk factors for surgery include worse visual acuity at presentation (20/60 Snellen visual acuity in surgical patients versus 20/40 Snellen visual acuity in nonsurgical patients, P<0.001, greater average central corneal thickness (635 µm versus 592 µm, P<0.001, loss of visual acuity over time (two lines lost versus zero lines lost, P<0.001, increasing age (P<0.001, and male sex (P=0.008. Over half of patients (52% did not receive surgery despite poor vision. Conclusion: During this time period, FECD did not have a consistent pattern for management or treatment, and despite

  18. [Treatment of corneal endothelial disorders by DMEK and UT-DSAEK. Indications, complications, results and follow-up].

    Science.gov (United States)

    Bachmann, B; Schaub, F; Cursiefen, C

    2016-03-01

    Various techniques for posterior lamellar keratoplasty have been established for the clinical routine and continuously improved during the last 15 years so that an extremely rapid recovery of vision is possible due to very thin transplants. Descemet membrane endothelial keratoplasty (DMEK) is the method of choice for simple corneal endothelial diseases and has already been applied in complex conditions of the anterior segment. The learning curve for DMEK is comparatively long and the risk of complications in complex anterior segment pathologies is higher than in Descemet's stripping automated endothelial keratoplasty (DSAEK); however, DMEK results in better visual outcome and less graft rejections than DSAEK. The latest evolution in posterior lamellar transplant surgery is ultrathin DSAEK (UT-DSAEK), where the grafted lamella is much thinner than in conventional DSAEK. Currently available data suggest that the resulting visual acuity after UT-DSAEK is close to the visual acuity seen after DMEK; however, studies comparing the results after DMEK and UT-DSAEK are so far lacking. Whether the transplantation of these very thin DSAEK grafts also results in endothelial cell densities and graft rejection rates comparable to DMEK has to be proven.

  19. Toxic endothelial cell destruction of the cornea after routine extracapsular cataract surgery.

    Science.gov (United States)

    Breebaart, A C; Nuyts, R M; Pels, E; Edelhauser, H F; Verbraak, F D

    1990-08-01

    Eighteen patients developed an acute corneal decompensation following normal intraocular surgery (cataract extraction in 17 patients), characterized by star-shaped endothelial folds, a twofold increase in corneal thickness, and a visual acuity of counting fingers during several postoperative days. In some cases, there was an additional iritis and transient hypotony. There was no effect of topical and/or subconjunctival corticosteroids on the course of the decompensation. Endothelial morphometric analysis showed a mean endothelial cell loss of 72%. Endothelial wound healing, as determined by coefficient of variation and percentage hexagonals, stabilized 6 months postoperatively. We coined the term toxic endothelial cell destruction for this syndrome. Epidemiological evaluation revealed the toxic endothelial cell destruction syndrome to be linked with the 10-fold increase of a detergent solution in the ultrasonic bath for cleaning the surgical instruments.

  20. Corneal endothelial rejection after penetrating keratoplasty treated with intravenous and topic corticosteroid: one year follow up

    Directory of Open Access Journals (Sweden)

    Ricardo Yuji Abe

    2013-02-01

    Full Text Available OBJECTIVE: To analyze the recovery of visual acuity (VA and graft survival after first episode of endothelial rejection in penetrating keratoplasty (PKP treated with intravenous (IV and topic corticosteroid. METHODS: Interventional, prospective, non-comparative case series study evolving 32 PKP patients in one year follow up, who presented first episode of corneal endothelial rejection. The patients were submitted to 500 mg IV injection of methylprednisolone in association with topical prednisolone. Main outcome measures included VA recovery and corneal edema regression. Second outcome included new rejections and graft failure. Multivariate analysis techniques were used to estimate rates of graft outcome events and the impact of risk factors. RESULTS: A total of 32 eyes from 32 patients (13 male and 19 female were included in the study. The mean VA (in number of letters before rejection was 48 (22 to 88 letters. Patients treated within 7 days or less of initial symptoms had better VA recovery, corneal edema regression and less graft failure (p<0.001. Patients with previous ocular surgery had worse VA recovery and more graft failure (p<0.047. CONCLUSION: The association between the other risk factors and the outcomes did not reach statistical significance in the multivariate model because of the small numbers of patients. Methylprednisolone in association with topical prednisolone is an alternative treatment for graft rejection. Our study showed that patients treated within 7 days of symptoms and no previous anterior segment surgery had better visual outcome and graft survival after treatment.

  1. A multicenter study to map genes for Fuchs endothelial corneal dystrophy: baseline characteristics and heritability.

    Science.gov (United States)

    Louttit, Megan D; Kopplin, Laura J; Igo, Robert P; Fondran, Jeremy R; Tagliaferri, Angela; Bardenstein, David; Aldave, Anthony J; Croasdale, Christopher R; Price, Marianne O; Rosenwasser, George O; Lass, Jonathan H; Iyengar, Sudha K

    2012-01-01

    To describe the methods for family and case-control recruitment for a multicenter genetic and associated heritability analyses of Fuchs endothelial corneal dystrophy (FECD). Twenty-nine enrolling sites with 62 trained investigators and coordinators gathered individual and family information, graded the phenotype, and collected blood and/or saliva for genetic analysis on all individuals with and without FECD. The degree of FECD was assessed in a 0 to 6 semiquantitative scale using standardized clinical methods with pathological verification of FECD on at least 1 member of each family. Central corneal thickness was measured by ultrasonic pachymetry. Three hundred twenty-two families with 330 affected sibling pairs with FECD were enrolled and included a total of 650 sibling pairs of all disease grades. Using the entire 7-step FECD grading scale or a dichotomous definition of severe disease, heritability was assessed in families via sib-sib correlations. Both binary indicators of severe disease and semiquantitative measures of disease severity were significantly heritable, with heritability estimates of 30% for severe disease, 37% to 39% for FECD score, and 47% for central corneal thickness. Genetic risk factors have a strong role in the severity of the FECD phenotype and corneal thickness. Genotyping this cohort with high-density genetic markers followed by appropriate statistical analyses should lead to novel loci for disease susceptibility.

  2. 房水培养对牛角膜内皮细胞生长的影响%Influence of aqueous humor on growth of bovine corneal endothelial cell in vitro

    Institute of Scientific and Technical Information of China (English)

    李善义; 戴应; 谭美华; 丁勇; 钟敬祥; 陈建苏

    2013-01-01

    Background The construction of tissue-engineered corneal endothelium needs the functional seeding cells,so how to culture a large amount of functional corneal endothelial cells (CECs) is an urgent problem to be solved.Objective The aim of this study was to evaluate the role of aqueous humor on bovine CECs in vitro.Methods Aqueous humor of 1.2 ml was collected from the anterior chamber of bovine and sterilized,and the liquid supernatant was obtained.The bovine CECs were isolated from bovine cornea and then cultured in low glucose Dulbecco Modified Eagle Medium with 10% fetal bovine serum (FBS) in vitro.Aqueous humor was added into the medium with the final concentration of 2.5%,5.0%,l0.0%,15.0% and 20.0%,respectively,and no aqueous humor was added in the control group.Cell counting kit-8 (CCK-8) assay was used to detect the absorbency value of CECs for the evaluation of cell proliferation.Progression of the cell cycle was analyzed by flow cytometry (FCM).After confluence of the cells was reached,1 ml plastic spear tip was used to scratch the cell single layer,and the cells were incubated consequently in medium with 10% FBS and with or without aqueous humor for 24 hours.Healing area of the cell single layer was measured.The cells were incubated at a density of 6 × 105 cells/ml and cultured using medium with or without 10.0% aqueous human for 5 days,and the number of the cells was analyzed by DAPI fluorescence technique.Results Under the phase-contrast microscopy,the confluent CECs showed a slabstone-like and hexagonal appearance.CCK-8 assay revealed that the absorbance values of CECs was significantly different among the various culture groups (F=4.051,P =0.007),and the absorbance value in different concentrations of aqueous human culture groups was significantly higher than that in the control group (P < 0.01).FCM showed that the percentage of the cells in S-G2 phases was (34.80-±3.13)% in the 10.0% aqueous humors group and (23.06±1.13)

  3. Isolation and culture of corneal neovascular endothelial cells and expression of chemokine receptors%小鼠角膜新生血管内皮细胞的分离培养及其趋化因子受体的表达

    Institute of Scientific and Technical Information of China (English)

    刘高勤; 肖艳辉; 陈志刚; 徐静; 陆培荣

    2016-01-01

    Background The pathogenesis and mechanism research of corneal neovascularization is of important significance for the prevention and management of corneal neovascularization.Some relative researches are being performed on non-corneal neovascularization-derived vascular endothelial cells, so the results are affected to a certain extent.Objective This study was to isolate and culture vascular endothelial cells from experimental corneal neovascularization tissue and detect the expression of chemokine receptors in vitro.Methods Corneal neovascularization models were established on 10 SPF male BALB/c mice with the age of 7-8 weeks by sticking the filter papers with NaOH on the central corneas, and then the immunofluorescence technique was use to assay the CD31 expression in corneal flatmount 2 weeks after modeling.Corneal pieces were made in 2 weeks after alkali burn and then were digested by collagenase type D.Vascular endothelial cells were isolated from neovascularized tissue by affinity purification using magnetic beads coated with anti-CD31.The cells were cultured on fibronectin-coated walls and then identified by immunocytochemistry.Reverse transcription-PCR was employed to detect the expressions of chemokine receptors in the cells.The use and care of the animals complied with ARVO Statement and this experimental procedure was approved by Soochow University Animal Care Committee.Results Corneal neovascularization occurred at 7 days and peaked at 2 weeks after modeling, and immunofluorescence exhibited the green network-like fluorescence for CD31 antibody in corneas.The cells grew against the wall 2 hours after culture with the polygon shape and large dimension, and the growth obviously quickened after passage.The cultured cells showed the positive response for CD31 antibody, showing the brown dye in cytoplasm,in contrast,the expression of CD31 was absent in corneal stromal cells.Chemokine receptors were positively expressed in the cells with the strongest

  4. Promotive effect of platelet-rich plasma on proliferation of corneal endothelial cells in cats%富血小板血浆促猫角膜内皮细胞增殖的研究

    Institute of Scientific and Technical Information of China (English)

    王新法; 徐锦堂

    2011-01-01

    目的 观察富血小板血浆对体外培养猫角膜内皮细胞增殖的影响.方法 采用揭膜法与消化法相结合获取猫角膜原代内皮细胞,培养基采用DMEM培养基(含体积分数10%胎牛血清).采用二次离心法提取富血小板血浆.在完全培养基中加入体积分数分别为5%、10%、20%的富血小板血浆培养角膜内皮细胞,以未加入富血小板血浆的完全培养基为对照.CCK-8试剂盒检测细胞增殖情况,扫描电镜观察细胞形态变化.结果 富血小板血浆体外培养角膜内皮细胞时间越长,其促增殖作用越明显,与对照组比较差异均有显著统计学意义意义(均为P <0.01),且随着富血小板血浆含量的增加,角膜内皮细胞增殖越明显,呈剂量依赖性.扫描电镜检测表明,与对照组相比,PRP作用组角膜内皮细胞表面可见丰富的微绒毛,且随着富血小板血浆含量的增加,微绒毛越丰富.结论 富血小板血浆能明显促进体外培养猫角膜内皮细胞增殖.%Objective To observe effect of platelet-rich plasma (PRP) on proliferation of corneal endothelial cells (CEC) in cats in vitro. Methods The cat CEC were obtained by separating and treating with enzyme digestion,then cultured in DMEM supplemented with 10% volume fractions fetal bovine serum (FBS). PRP was extracted by the two-step centrifugation method. The CEC of the second generation were cultured in the complete media containing different concentrations of PRP (5% , 10% and 20% volume fractions),while those cultured in the media without PRP were served as controls. Proliferation of CEC was detected by CCK-8 kit method. The morphological changes of CEC were examined by scanning electron microscope (SEM). Results Effect of proliferation of the CEC cultured with PRP was obvious with time prolonged, and had statistical difference compared with that of control group (P < 0. 01). The proliferation of CEC was more obvious with the increase of platelet

  5. EXPERIMENTAL STUDY ON THE CORNEAL ENDOTHELIUM OF TRAUMATIC CATARACT

    Institute of Scientific and Technical Information of China (English)

    1991-01-01

    The cell morphology of corneal endothelium in 84 mice with experimental traumatic cataract was investigated with stained corneal buttons. In the experimental group, the boundaries between adjacent corneal endothelial cells were significantly distorted, some cell boundaries manifested degenerative changes that led to coalescence of the cells. The mean density and mean area of endothelial cells of the controls showed significant difference from those of the experimental group during the 12 weeks of observ...

  6. Inhibition of corneal neovascularization with new Tyrosine Kinase Inhibitors targeting vascular endothelial growth factor receptors: Sunitinib malate and Sorafenib

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    Delnia Arshadi

    2007-06-01

    Full Text Available Corneal neovascularization (NV is a significant, sight-threatening, complication of many ocular surface disorders. Presence of new vessels in cornea can compromise clarity and thus vision. The data supporting a causal role for vascular endothelial growth factor (VEGF in corneal NV are extensive. Inhibition of VEGF remains as a main strategy for treating corneal NV. There is a growing body of evidence that corneal NV can be reduced by using anti-VEGF agents. Sunitinib malate and Sorafenib are new orally bio-available anti-angiogenic agents undergoing tests of efficacy in the treatment of various types of cancers. The main mechanism of these drugs is inhibiting angiogenesis by diminishing signaling through VEGF receptor1 (VEGFR1, VEGFR2, and platelet-derived growth factor receptors. Since VEGF exerts its angiogenic effects through tyrosine kinase receptors in cornea, any mechanisms which reduce VEGF signaling may inhibit corneal NV or at least attenuate it. Based on this fact we herein hypothesize that Sunitinib malate and Sorafenib can be prepared in topical form and be used in corneal neovascularization states. These approaches offer new hope for the successful treatment of corneal NV. Further investigations in animal models are needed to place these two drugs alongside corneal NV therapeutics.

  7. Estudo comparativo da densidade de células endoteliais da córnea após facoemulsificação pelas técnicas de "dividir e conquistar" e "quick chop" Corneal endothelial cell density comparative study after phacoemulsification by "divide and conquer" and "quick chop" techniques

    Directory of Open Access Journals (Sweden)

    Flavia Guedes Pinto Domingues

    2005-02-01

    Full Text Available OBJETIVOS: Uma vez que um dos fatores mais implicados na perda celular endotelial corneana após facoemulsificação é o tempo utilizado de ultra-som e que a técnica de "quick chop" utiliza menor tempo de ultra-som, o objetivo do estudo foi comparar e analisar a redução da densidade celular endotelial corneana entre os pacientes submetidos à facoemulsificação pelas técnicas de "dividir e conquistar" e "quick chop". MÉTODOS: A amostra do trabalho foi constituída por 56 pacientes, apresentando catarata senil grau 3, selecionados prospectivamente e submetidos à facoemulsificação endocapsular, através das técnicas de "dividir e conquistar" (28 pacientes, grupo 1 e "quick chop" (28 pacientes, grupo 2. Foram verificados os tempos de ultra-som utilizados em cada cirurgia e foram realizados exames de microscopia especular central de não-contato da córnea pré-operatórios e pós-operatórios de 1 mês, 3 meses e 6 meses. RESULTADOS: Verificamos que a técnica de "dividir e conquistar" utilizou em média 2,0 minutos de tempo de ultra-som e a de "quick chop" 1,1 minuto; que a redução da densidade celular endotelial central da córnea foi significativa, em ambas as técnicas, no primeiro mês pós-operatório (16,5% no grupo 1 e 19,4% no grupo 2; e que as variações de densidade celular endotelial corneana subseqüentes, aos 3 e 6 meses, não foram significativas. Não houve diferença estatística na variação da densidade celular endotelial corneana ocorrida entre os grupos (teste "t" de Student p=0,334; no 1º mês pós-operatório. CONCLUSÕES: Concluímos que a redução da densidade endotelial corneana central não foi estatisticamente diferente entre as técnicas.PURPOSE: Since one of the most implicated factors in corneal endothelial cell loss after phacoemulsification is the ultrasound time, and that the "quick chop" technique utilizes less ultrasound time, the objective of this study was to compare and analyze the reduction

  8. Combining femtosecond laser ablation and diode laser welding in lamellar and endothelial corneal transplants

    Science.gov (United States)

    Pini, Roberto; Rossi, Francesca; Matteini, Paolo; Ratto, Fulvio; Menabuoni, Luca; Lenzetti, Ivo; Yoo, Sonia H.; Parel, Jean-Marie

    2008-02-01

    Based on our previous clinical experiences in minimally invasive diode laser-induced welding of corneal tissue in penetrating keratoplasty (PK), i.e. full-thickness transplant of the cornea, we combined this technique with the use of a femtosecond laser for applications in lamellar (LK) and endothelial (EK) keratoplasty. In LK, the femtosecond laser was used to prepare donor button and recipient corneal bed; the wound edges were stained with a water solution of Indocyanine Green (ICG) and then irradiated with a diode laser emitting in CW mode to induce stromal welding. Intraoperatory observations and follow-up results up to 6 months indicated the formation of a smooth stromal interface, total absence of edema as well as inflammation, and reduction of post-operative astigmatism, as compared with conventional suturing procedures. In EK the femtosecond laser was used for the preparation of a 100 μm thick, 8.5mm diameter donor corneal endothelium flap. The flap stromal side was stained with ICG. After stripping the recipient Descemet's membrane and endothelium, the donor flap was positioned in the anterior chamber on the inner face of the cornea by an air bubble and secured to the recipient cornea by diode laser pulses delivered by means of a fiberoptic contact probe introduced in the anterior chamber, which produced welding spots of 200 μm diameter. Femtosecond laser sculpturing of the donor cornea provided lamellar and endothelial flaps of preset and constant thickness. Diode laserinduced welding showed a unique potential to permanently secure the donor flap in place, avoiding postoperative displacement and inflammation reaction.

  9. Comparison of morphological and functional endothelial cell changes after cataract surgery: Phacoemulsification versus manual small-incision cataract surgery

    Directory of Open Access Journals (Sweden)

    Sunil Ganekal

    2014-01-01

    Conclusion: The central corneal thickness, coefficient of variation, and standard deviation were maintained in both groups indicating that the function and morphology of endothelial cells was not affected despite an initial reduction in endothelial cell number in MSICS. Thus, MSICS remains a safe option in the developing world.

  10. Effects of timolol on bovine corneal endothelial cultures.

    Science.gov (United States)

    Staatz, W D; Radius, R L; Van Horn, D L; Schultz, R O

    1981-04-01

    The chronic use of timolol (Timoptic) maleate to control glaucoma may produce cytotoxic complications in the cornea. We have therefore compared the relative toxic effects of the commercial ophthalmic preparation with that of the pure compound. Commercial vehicle, either with or without 16 mM timolol maleate, killed cultures within the first five minutes of exposure. Pure timolol maleate, however, caused rapid but reversible cellular contractions, and cells remained viable in it for over 24 hours. Dilution with culture medium reduced both the cytotoxicity and the speed of the contractions. Incubation in 1:100 dilutions of vehicle or commercial drug preparations or in 0.16 mM pure timolol maleate did not alter cellular morphology. The results indicate that while undiluted vehicle is toxic, timolol maleate is not.

  11. Generation of corneal epithelial cells from induced pluripotent stem cells derived from human dermal fibroblast and corneal limbal epithelium.

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    Ryuhei Hayashi

    Full Text Available Induced pluripotent stem (iPS cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF-derived iPS cells (253G1 and human adult corneal limbal epithelial cells (HLEC-derived iPS cells (L1B41. We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA differentiation method, as Pax6(+/K12(+ corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.

  12. Limbal stem cells: Central concepts of corneal epithelial homeostasis

    Institute of Scientific and Technical Information of China (English)

    Jinny; J; Yoon; Salim; Ismail; Trevor; Sherwin

    2014-01-01

    A strong cohort of evidence exists that supports the localisation of corneal stem cells at the limbus. The distinguishing characteristics of limbal cells as stem cells include slow cycling properties, high proliferative potential when required, clonogenicity, absence of differentiation marker expression coupled with positive expression of progenitor markers, multipotency, centripetal migration, requirement for a distinct niche environment and the ability of transplanted limbal cells to regenerate the entire corneal epithelium. The existence of limbal stem cells supports the prevailing theory of corneal homeostasis, known as the XYZ hypothesis where X represents proliferation and stratification of limbal basal cells, Y centripetal migration of basal cells and Z desquamation of superficial cells. To maintain the mass of cornea, the sum of X and Y must equal Z and very elegant cell tracking experiments provide strong evidence in support of this theory. However, several recent stud-ies have suggested the existence of oligopotent stem cells capable of corneal maintenance outside of the limbus. This review presents a summary of data which led to the current concepts of corneal epithelial homeostasis and discusses areas of controversy surrounding the existence of a secondary stem cell reservoir on the corneal surface

  13. Cell pattern in adult human corneal endothelium.

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    Carlos H Wörner

    Full Text Available A review of the current data on the cell density of normal adult human endothelial cells was carried out in order to establish some common parameters appearing in the different considered populations. From the analysis of cell growth patterns, it is inferred that the cell aging rate is similar for each of the different considered populations. Also, the morphology, the cell distribution and the tendency to hexagonallity are studied. The results are consistent with the hypothesis that this phenomenon is analogous with cell behavior in other structures such as dry foams and grains in polycrystalline materials. Therefore, its driving force may be controlled by the surface tension and the mobility of the boundaries.

  14. In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Ken Kono

    Full Text Available Functionalizing biomaterials with peptides or polymers that enhance recruitment of endothelial cells (ECs can reduce blood coagulation and thrombosis. To assess endothelialization of materials in vitro, primary ECs are generally used, although the characteristics of these cells vary among the donors and change with time in culture. Recently, primary cell lines immortalized by transduction of simian vacuolating virus 40 large T antigen or human telomerase reverse transcriptase have been developed. To determine whether immortalized ECs can substitute for primary ECs in material testing, we investigated endothelialization on biocompatible polymers using three lots of primary human umbilical vein endothelial cells (HUVEC and immortalized microvascular ECs, TIME-GFP. Attachment to and growth on polymer surfaces were comparable between cell types, but results were more consistent with TIME-GFP. Our findings indicate that TIME-GFP is more suitable for in vitro endothelialization testing of biomaterials.

  15. Comparison of corneal endothelial changes following phacoemulsification with transversal and torsional phacoemulsification machines

    Institute of Scientific and Technical Information of China (English)

    Mustafa; Atas; Süleyman; Demircan; Arzu; Seyhan; Karatepe; Hashas; Ahmet; Gülhan; Gkmen; Zararsιz

    2014-01-01

    AIM:To compare and evaluate the phacoemulsification parameters and postoperative endothelial cell changes of two different phacoemulsification machines, each with different modes, but also to assess the relationship between postoperative endothelial cell loss and the phacoemulsification parameters, as well as the other factors in both groups.METHODS:This prospective observational study was comprised of consecutive eligible cataract patients operated with phacoemulsification technique performed by the same surgeon using either a WHITESTAR Signature Ellips FX(transversal, group 1) or Infiniti OZil IP(torsional, group 2) machine.RESULTS:The study included 86 patients. Baseline characteristics in the groups were similar. The median nuclear sclerosis grade was 3(2-4) in the first group and2(2-4) in the second group(P =0.265). Both groups had similar phacoemulsification needle times(group 1: 60.63±36 s; group 2: 55.98±30 s; P =0.789). The percentage of endothelial cell loss 30 d after surgery ranged from 3% to15% with a median of 7% in group 1, and from 2% to13% with a median of 6% in group 2; however, there was no statistically significant difference between the groups(P =0.407). Hexagonality(P =0.794) and the coefficient of variation(CV; P =0.142) did not differ significantly between the groups before and 30 d after surgery. Asignificant positive correlation was found between the endothelial cell loss and nuclear sclerosis grade(group1: P <0.001; group 2: P <0.001) and between the endothelial cell loss and average phacoemulsification power(group 1: P =0.007; group 2: P =0.008).CONCLUSION:Both of these machines were efficient,with similar endothelial cell loss. This endothelial cell loss was related to the increased nuclear sclerosis grade and increased phacoemulsification power.

  16. VISUAL PERCEPTION BASED AUTOMATIC RECOGNITION OF CELL MOSAICS IN HUMAN CORNEAL ENDOTHELIUMMICROSCOPY IMAGES

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    Yann Gavet

    2011-05-01

    Full Text Available The human corneal endothelium can be observed with two types of microscopes: classical optical microscope for ex-vivo imaging, and specular optical microscope for in-vivo imaging. The quality of the cornea is correlated to the endothelial cell density and morphometry. Automatic methods to analyze the human corneal endothelium images are still not totally efficient. Image analysis methods that focus only on cell contours do not give good results in presence of noise and of bad conditions of acquisition. More elaborated methods introduce regional informations in order to performthe cell contours completion, thus implementing the duality contour-region. Their good performance can be explained by their connections with several basic principles of human visual perception (Gestalt Theory and Marr's computational theory.

  17. Effect of substrate composition and alignment on corneal cell phenotype.

    Science.gov (United States)

    Phu, Donna; Wray, Lindsay S; Warren, Robert V; Haskell, Richard C; Orwin, Elizabeth J

    2011-03-01

    Corneal blindness is a significant problem treated primarily by corneal transplants. Donor tissue supply is low, creating a growing need for an alternative. A tissue-engineered cornea made from patient-derived cells and biopolymer scaffold materials would be widely accessible to all patients and would alleviate the need for donor sources. Previous work in this lab led to a method for electrospinning type I collagen scaffolds for culturing corneal fibroblasts ex vivo that mimics the microenvironment in the native cornea. This electrospun scaffold is composed of small-diameter, aligned collagen fibers. In this study, we investigate the effect of scaffold nanostructure and composition on the phenotype of corneal stromal cells. Rabbit-derived corneal fibroblasts were cultured on aligned and unaligned collagen type I fibers ranging from 50 to 300 nm in diameter and assessed for expression of α-smooth muscle actin, a protein marker upregulated in hazy corneas. In addition, the optical properties of the cell-matrix constructs were assessed using optical coherence microscopy. Cells grown on collagen scaffolds had reduced myofibroblast phenotype expression compared to cells grown on tissue culture plates. Cells grown on aligned collagen type I fibers downregulated α-smooth muscle actin protein expression significantly more than unaligned collagen scaffolds, and also exhibited reduced overall light scattering by the tissue construct. These results suggest that aligned collagen type I fibrous scaffolds are viable platforms for engineering corneal replacement tissue.

  18. Peripheral endothelial cell damage after trephination of donor tissue.

    Science.gov (United States)

    Terry, Mark A; Saad, Hisham A; Shamie, Neda; Shah, Anand K

    2009-12-01

    To evaluate and quantify the degree and pattern of donor endothelial cell damage, which occurs with mechanical trephination of donor corneal tissue. Twenty donor corneal-scleral tissues were used for these paired experiments. The tissues were randomized for trephination with 10 tissues trephinated by an 8.0-mm-diameter Barron trephine (Katena, Denville, NJ), and 10 tissues trephinated with an 8.0-mm-diameter UltraFit Coronet trephine (distributed by Angiotech, British Columbia, Canada) by the same investigator. Trephinated corneal buttons were then stained with vital dye stain, and the endothelial layer image captured with digital photography. The images were then analyzed by digital planimetry, and the pattern and quantity of endothelial damage was determined by an investigator who was masked to the specific trephine used for the individual tissue. Trephination created a pattern of circular damage at the edge of the donor button in every case with no break in continuity of the circle, but some portions of the circle were wider than others. Occasional, scattered, peripheral small areas also displayed damage, but no significant striae, stretch, or other central damage was noted in any donor. The mean percent damage in the series was 6.35% +/- 0.90% (range: 4.33%-7.78%). The UltraFit Coronet trephinations averaged damage of 5.64% +/- 0.85% (range: 4.33%-6.69%), and the Barron trephinations averaged damage of 6.50% +/- 0.95% (range: 4.92%-7.78%). Although 8 of 10 experimental pairs of trephinations demonstrated less peripheral endothelial damage with the UltraFit Coronet trephine, the mean damage between each group did not reach statistical significance in this small series. (P = 0.08) Donor mechanical trephination of full-thickness corneal tissue creates relatively consistent amounts of peripheral edge damage and likely no central endothelial damage. There may exist differences in edge damage between different mechanical trephination systems, and a direct comparison

  19. Oxidized alginate hydrogels as niche environments for corneal epithelial cells.

    Science.gov (United States)

    Wright, Bernice; De Bank, Paul A; Luetchford, Kim A; Acosta, Fernando R; Connon, Che J

    2014-10-01

    Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P ≤ 0.05) and this improved further with addition of collagen IV (P ≤ 0.01). Oxidised gels presented larger internal pores (diameter: 0.2-0.8 µm) than unmodified gels (pore diameter: 0.05-0.1 µm) and were significantly less stiff (P ≤ 0.001), indicating that an increase in pore size and a decrease in stiffness contributed to improved cell viability. The diffusion of collagen IV from oxidised alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy.

  20. Oxidized alginate hydrogels as niche environments for corneal epithelial cells

    Science.gov (United States)

    Wright, Bernice; De Bank, Paul A; Luetchford, Kim A; Acosta, Fernando R; Connon, Che J

    2014-01-01

    Chemical and biochemical modification of hydrogels is one strategy to create physiological constructs that maintain cell function. The aim of this study was to apply oxidised alginate hydrogels as a basis for development of a biomimetic niche for limbal epithelial stem cells that may be applied to treating corneal dysfunction. The stem phenotype of bovine limbal epithelial cells (LEC) and the viability of corneal epithelial cells (CEC) were examined in oxidised alginate gels containing collagen IV over a 3-day culture period. Oxidation increased cell viability (P ≤ 0.05) and this improved further with addition of collagen IV (P ≤ 0.01). Oxidised gels presented larger internal pores (diameter: 0.2–0.8 µm) than unmodified gels (pore diameter: 0.05–0.1 µm) and were significantly less stiff (P ≤ 0.001), indicating that an increase in pore size and a decrease in stiffness contributed to improved cell viability. The diffusion of collagen IV from oxidised alginate gels was similar to that of unmodified gels suggesting that oxidation may not affect the retention of extracellular matrix proteins in alginate gels. These data demonstrate that oxidised alginate gels containing corneal extracellular matrix proteins can influence corneal epithelial cell function in a manner that may impact beneficially on corneal wound healing therapy. © 2013 The Authors. Journal of Biomedical Materials Research Part A Published byWiley Periodicals, Inc. Part A: 102A: 3393–3400, 2014. PMID:24142706

  1. 以猪角膜脱细胞基质为载体培养人脐静脉内皮细胞构建实验性角膜后板层%Decellularized porcine corneal posterior lamellae as carrier matrix for cultivating human umbilical vein endothelial cells in vitro

    Institute of Scientific and Technical Information of China (English)

    祁冰; 侯光辉; 李柳; 季青山; 吴静; 周清

    2013-01-01

    AIM:To investigate the feasibility of corneal posterior lamellar reconstruction with human umbilical vein endothelial cells (HUVECs) and porcine cornea acellular matrix in vitro,and to observe the physiological function of the transplantation in vivo.METHODS:HUVECs were isolated,cultured,and labeled with fluorescent dye CM-DiI.Porcine corneas were treated with 100% glycerinum,cut to a thinner structure step by step,and dried on the super-clean bench.Transmission electron microscope were used to observe the histological changes of the porcine cornes acellular matrix.Labeled HUVECs were seeded onto the porcine cornea acellular matrix,and examined by scanning electron microscopy.When the HUVECs and Descemet's membrane fusion formed a monolayer,the corneal transplantation in rabbits was performed.Twenty-four New Zealand white rabbits were randomly divided into experimental group and control group (n =12 each),and their left eyes served as recipients.RESULTS:Cultured HUVECs exhibited polygonal shape.More than 90% HUVECs were labeled with CM-DiI and the cell membrane was positive with red fluorescence,which was detectable at least up to 3 generations.The histological examination indicated that porcine cornea cells were clearly extracted,and the collagen fibers were well arranged.A continuous monolayer of HUVECs on the porcine cornea acellular matrix was observedunder scanning electron microscopy.The reconstructed corneal posterior lamellae were similar to the noral cornea.The observation of transplantation showed that the cornea in experimental group was substantially transparent.However,that in control group was oedematous and adiaphanous.CONCLUSION:Corneal posterior lamellae can be reconstructedin vitro by cultivating HUVECs on porcine cornea acellular matrix.After xenogeneic transplantation,the graft survivesin vivo and expresses normal corneal endothelial cell biological functions.Deep lamellar corneal endothelial transplantation is an effective keratoplasty.%

  2. Trefoil peptides promote restitution of wounded corneal epithelial cells.

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    Göke, M N; Cook, J R; Kunert, K S; Fini, M E; Gipson, I K; Podolsky, D K

    2001-04-01

    The ocular surface shares many characteristics with mucosal surfaces. In both, healing is regulated by peptide growth factors, cytokines, and extracellular matrix proteins. However, these factors are not sufficient to ensure most rapid healing. Trefoil peptides are abundantly expressed epithelial cell products which exert protective effects and are key regulators of gastrointestinal epithelial restitution, the critical early phase of cell migration after mucosal injury. To assess the role of trefoil peptides in corneal epithelial wound healing, the effects of intestinal trefoil factor (ITF/TFF3) and spasmolytic polypeptide (SP/TFF2) on migration and proliferation of corneal epithelial cells were analyzed. Both ITF and SP enhanced restitution of primary rabbit corneal epithelial cells in vitro. While the restitution-enhancing effects of TGF-alpha and TGF-beta were both inhibited by neutralizing anti-TGF-beta-antibodies, trefoil peptide stimulation of restitution was not. Neither trefoil peptide significantly affected proliferation of primary corneal epithelial cells. ITF but not SP or pS2 mRNA was present in rabbit corneal and conjunctival tissues. In summary, the data indicate an unanticipated role of trefoil peptides in healing of ocular surface and demand rating their functional actions beyond the gastrointestinal tract.

  3. Differential expression of the Slc4 bicarbonate transporter family in murine corneal endothelium and cell culture.

    Science.gov (United States)

    Shei, William; Liu, Jun; Htoon, Hla M; Aung, Tin; Vithana, Eranga N

    2013-01-01

    To characterize the relative expression levels of all the solute carrier 4 (Slc4) transporter family members (Slc4a1-Slc4a11) in murine corneal endothelium using real-time quantitative (qPCR), to identify further important members besides Slc4a11 and Slc4a4, and to explore how close to the baseline levels the gene expressions remain after cells have been subjected to expansion and culture. Descemet's membrane-endothelial layers of 8-10-week-old C57BL6 mice were stripped from corneas and used for both primary cell culture and direct RNA extraction. Total RNA (from uncultured cells as well as cultured cells at passages 2 and 7) was reverse transcribed, and the cDNA was used for real time qPCR using specific primers for all the Slc4 family members. The geNorm method was applied to determine the most stable housekeeping genes and normalization factor, which was calculated from multiple housekeeping genes for more accurate and robust quantification. qPCR analyses revealed that all Slc4 bicarbonate transporter family members were expressed in mouse corneal endothelium. Slc4a11 showed the highest expression, which was approximately three times higher than that of Slc4a4 (3.4±0.3; p=0.004). All Slc4 genes were also expressed in cultured cells, and interestingly, the expression of Slc4a11 in cultured cells was significantly reduced by approximately 20-fold (0.05±0.001; p=0.000001) in early passage and by approximately sevenfold (0.14±0.002; p=0.000002) in late passage cells. Given the known involvement of SLC4A4 and SLC4A11 in corneal dystrophies, we speculate that the other two highly expressed genes in the uncultured corneal endothelium, SLC4A2 and SLC4A7, are worthy of being considered as potential candidate genes for corneal endothelial diseases. Moreover, as cell culture can affect expression levels of Slc4 genes, caution and careful design of experiments are necessary when undertaking studies of Slc4-mediated ion transport in cultured cells.

  4. A Multi-Center Study to Map Genes for Fuchs’ Endothelial Corneal Dystrophy: Baseline Characteristics and Heritability

    Science.gov (United States)

    Louttit, Megan D; Kopplin, Laura J; Igo, Robert P; Fondran, Jeremy R; Tagliaferri, Angela; Bardenstein, David; Aldave, Anthony J; Croasdale, Christopher R; Price, Marianne; Rosenwasser, George O; Lass, Jonathan H; Iyengar, Sudha K

    2013-01-01

    Purpose To describe the methods for family and case-control recruitment for a multi-center genetic and associated heritability analysis of Fuchs’ Endothelial Corneal Dystrophy (FECD). Methods Twenty-nine enrolling sites with 62 trained investigators and coordinators gathered individual and family information, graded the phenotype, and collected blood and/or saliva for genetic analysis on all individuals with and without FECD. The degree of FECD was assessed in a 0–6 semi-quantitative scale using standardized clinical methods with pathologic verification of FECD on at least one member of each family. Central corneal thickness was measured by ultrasonic pachymetry. Results Three hundred twenty-two families with 330 affected sibling pairs with FECD were enrolled, and included a total of 650 sibling pairs of all disease grades. Using the entire 0–6 step FECD grading scale or a dichotomous definition of severe disease, heritability was assessed in families via sib-sib correlations. Both binary indicators of severe disease as well as semi-quantitative measures of disease severity were significantly heritable, with heritability estimates of 30% for severe disease, 37–39% for FECD score and 47% for central corneal thickness. Conclusion Genetic risk factors have a strong role in the severity of the FECD phenotype and corneal thickness. Genotyping this cohort with high-density genetic markers followed by appropriate statistical analyses should lead to novel loci for disease susceptibility. PMID:22045388

  5. Derivation of Corneal Keratocyte-Like Cells from Human Induced Pluripotent Stem Cells

    Science.gov (United States)

    Naylor, Richard W.; McGhee, Charles N. J.; Cowan, Chad A.; Davidson, Alan J.; Holm, Teresa M.; Sherwin, Trevor

    2016-01-01

    Corneal diseases such as keratoconus represent a relatively common disorder in the human population. However, treatment is restricted to corneal transplantation, which only occurs in the most advanced cases. Cell based therapies may offer an alternative approach given that the eye is amenable to such treatments and corneal diseases like keratoconus have been associated specifically with the death of corneal keratocytes. The ability to generate corneal keratocytes in vitro may enable a cell-based therapy to treat patients with keratoconus. Human induced pluripotent stem cells (hiPSCs) offer an abundant supply of cells from which any cell in the body can be derived. In the present study, hiPSCs were successfully differentiated into neural crest cells (NCCs), the embryonic precursor to keratocytes, and then cultured on cadaveric corneal tissue to promote keratocyte differentiation. The hiPSC-derived NCCs were found to migrate into the corneal stroma where they acquired a keratocyte-like morphology and an expression profile similar to corneal keratocytes in vivo. These results indicate that hiPSCs can be used to generate corneal keratocytes in vitro and lay the foundation for using these cells in cornea cell-based therapies. PMID:27792791

  6. Circulating Endothelial Cells and Endothelial Progenitor Cells in Pediatric Sepsis.

    Science.gov (United States)

    Zahran, Asmaa Mohamad; Elsayh, Khalid Ibrahim; Mohamad, Ismail Lotfy; Hassan, Gamal Mohamad; Abdou, Madleen Adel A

    2016-03-01

    The aim of the study was to measure the number of circulating endothelial cells (CECs) and circulating endothelial progenitor cells (CEPs) in pediatric patients with sepsis and correlating it with the severity of the disease and its outcome. The study included 19 children with sepsis, 26 with complicated sepsis, and 30 healthy controls. The patients were investigated within 48 hours of pediatric intensive care unit admission together with flow cytometric detection of CECs and CEPs. The levels of both CECs and CEPs were significantly higher in patient with sepsis and complicated sepsis than the controls. The levels of CECs were higher in patients with complicated sepsis, whereas the levels of CEPs were lower in patients with complicated sepsis. Comparing the survival and nonsurvival septic patients, the levels of CEPs were significantly higher in the survival than in nonsurvival patients, whereas the levels of CECs were significantly lower in the survival than in nonsurvival patients. Serum albumin was higher in survival than in nonsurvival patients. Estimation of CECs and CEPs and their correlation with other parameters such as serum albumen could add important information regarding prognosis in septic pediatric patients.

  7. PPAR Gamma and Angiogenesis: Endothelial Cells Perspective

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    Jerzy Kotlinowski

    2016-01-01

    Full Text Available We summarize the current knowledge concerning PPARγ function in angiogenesis. We discuss the mechanisms of action for PPARγ and its role in vasculature development and homeostasis, focusing on endothelial cells, endothelial progenitor cells, and bone marrow-derived proangiogenic cells.

  8. Polymorphisms of the Homologous Recombination Gene RAD51 in Keratoconus and Fuchs Endothelial Corneal Dystrophy

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    Ewelina Synowiec

    2013-01-01

    Full Text Available Purpose. We investigated the association between genotypes and haplotypes of the c.-61G>T (rs 1801320 and c.-98G>C (rs 1801321 polymorphisms of the RAD51 gene and the occurrence of keratoconus (KC and Fuchs endothelial corneal dystrophy (FECD in dependence on some environmental factors. Methods. The polymorphisms were genotyped in peripheral blood lymphocytes of 100 KC and 100 FECD patients as well as 150 controls with PCR-RFLP. Results. The G/T genotype of the c.-61G>T polymorphism was associated with significantly increased frequency occurrence of KC (crude OR 2.99, 95% CI 1.75–5.13. On the other hand, the G/G genotype of this polymorphism was positively correlated with a decreased occurrence of this disease (crude OR 0.52, 95% CI 0.31–0.88. We did not find any correlation between genotypes/alleles of the c.-98G>C polymorphism and the occurrence of KC. We also found that the G/G genotype and G allele of the c.-98G>C polymorphism had a protective effect against FECD (crude OR 0.51, 95% CI 0.28–0.92; crude OR 0.53, 95% CI 0.30–0.92, resp., while the G/C genotype and the C allele increased FECD occurrence (crude OR 1.85, 95% CI 1.01–3.36; crude OR 1.90, 95% CI 1.09–3.29, resp.. Conclusions. The c.-61T/T and c.-98G>C polymorphisms of the RAD51 gene may have a role in the KC and FECD pathogenesis and can be considered as markers in these diseases.

  9. Genetic analysis of patients with Fuchs endothelial corneal dystrophy in India

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    Prajna Namperumalsamy V

    2010-02-01

    Full Text Available Abstract Background Mutations in COL8A2 gene which encodes the collagen alpha-2 (VIII chain have been identified in both familial and sporadic cases of Fuchs endothelial corneal dystrophy (FECD. Heterozygous mutations in the SLC4A11 gene are also known to cause late-onset FECD. Therefore we screened for COL8A2, SLC4A11 gene variants in Indian FECD patients. Methods Eighty patients with clinically diagnosed FECD and 100 age matched normal individuals were recruited. Genomic DNA was isolated from peripheral blood leukocytes. Mutations in COL8A2, SLC4A11 coding regions were screened using bi-directional sequencing. Fischer's exact test or Pearson's chi squared test were used to predict the statistical association of genotypes with the phenotype. Results Screening of COL8A2 gene revealed 2 novel c.1610G>A, c.1643A>G and 3 reported variations c.112G>A, c.464G>A and c.1485G>A. In SLC4A11 gene, novel c.1659C>T, c.1974C>T and reported c.405G>A, c.481A>C and c.639G>A variants were identified. However all the variations in both the genes were also present in unaffected controls. Conclusions This is the first study analysing COL8A2 gene in Indian patients with FECD. No pathogenic mutations were identified in COL8A2. Merely silent changes, which showed statistically insignificant association with FECD, were identified in the screening of SLC4A11 gene. These results suggest that COL8A2, SLC4A11 genes may not be responsible for FECD in patients examined in this study.

  10. Caveolin-1 associated adenovirus entry into human corneal cells.

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    Mohammad A Yousuf

    Full Text Available The cellular entry of viruses represents a critical area of study, not only for viral tropism, but also because viral entry dictates the nature of the immune response elicited upon infection. Epidemic keratoconjunctivitis (EKC, caused by viruses within human adenovirus species D (HAdV-D, is a severe, ocular surface infection associated with corneal inflammation. Clathrin-mediated endocytosis has previously been shown to play a critical role in entry of other HAdV species into many host cell types. However, HAdV-D endocytosis into corneal cells has not been extensively studied. Herein, we show an essential role for cholesterol rich, lipid raft microdomains and caveolin-1, in the entry of HAdV-D37 into primary human corneal fibroblasts. Cholesterol depletion using methyl-β-cyclodextrin (MβCD profoundly reduced viral infection. When replenished with soluble cholesterol, the effect of MβCD was reversed, allowing productive viral infection. HAdV-D37 DNA was identified in caveolin-1 rich endosomal fractions after infection. Src kinase activity was also increased in caveolin-1 rich endosomal fractions after infection, and Src phosphorylation and CXCL1 induction were both decreased in caveolin-1-/- mice corneas compared to wild type mice. siRNA knock down of caveolin-1 in corneal cells reduced chemokine induction upon viral infection, and caveolin-1-/- mouse corneas showed reduced cellular entry of HAdV-D37. As a control, HAdV-C2, a non-corneal pathogen, appeared to utilize the caveolar pathway for entry into A549 cells, but failed to infect corneal cells entirely, indicating virus and cell specific tropism. Immuno-electron microscopy confirmed the presence of caveolin-1 in HAdV-D37-containing vesicles during the earliest stages of viral entry. Collectively, these experiments indicate for the first time that HAdV-D37 uses a lipid raft mediated caveolin-1 associated pathway for entry into corneal cells, and connects the processes of viral entry with

  11. Elimination of Anterior Corneal Steepening With Descemet Membrane Endothelial Keratoplasty in a Patient With Fuchs Dystrophy and Keratoconus: Implications for IOL Calculation.

    Science.gov (United States)

    Gupta, Reena; Kinderyte, Ruta; Jacobs, Deborah S; Jurkunas, Ula V

    2017-10-01

    To report a case of coexistent Fuchs endothelial corneal dystrophy (FECD) and keratoconus (KCN) in which there was normalization of corneal topography after Descemet membrane endothelial keratoplasty (DMEK). Retrospective medical record review. Preoperative findings revealed a best-corrected visual acuity of 20/40 with -1.00 - 2.50 × 147, topographic maximum keratometry of 50.8 D with inferior steeping, and confluent guttae in the left eye. Medical record review revealed myopic shift, but little change in keratometry or corneal thickness over the previous 3 years. The patient developed epithelial edema with contact lens trial, highlighting endothelial dysfunction and eliminating the option of contact lenses for visual rehabilitation. Combined DMEK and cataract extraction with intraocular lens implantation was undertaken. Postoperatively, best-corrected visual acuity was 20/20 with only spherical correction. Elimination of stromal edema led to flattening of maximum anterior keratometry to 46.3 D and reduction of total corneal refractive power (TCRP) by 4.4 D. There was an unanticipated postoperative refractive error of +3.75 D consistent with this normalization of corneal topography. This is the first case report of the role of DMEK in normalizing corneal topography in coexistent FECD and KCN. The potential impact of DMEK on anterior curvature and TCRP must be considered in intraocular lens power calculation for cataract surgery in patients with FECD and KCN.

  12. 小鼠胚胎干细胞条件培养基体外促进人角膜内皮细胞增殖的研究%Proliferation in vitro of human corneal endothelial cells promoted by mouse embryonic stem cell conditioned medium

    Institute of Scientific and Technical Information of China (English)

    鹿晓燕

    2016-01-01

    目的 观察小鼠胚胎干细胞条件培养基(mouse embryonic stem cells conditioned medium,ESC-CM)是否可以在体外促进人角膜内皮细胞(human corneal endothelial cells,HCECs)的增殖.方法 利用角膜内皮后弹力层组织块方法进行原代培养P0 HCECs.实验组使用含有25% ESC-CM的培养液进行培养,对照组使用普通角膜内皮细胞培养液(corneal endothelium medium,CEM)进行培养.倒置相差显微镜、反转录聚合酶链反应(reverse-transcription polymerase chain reaction,RT-PCR)鉴定HCECs;倒置相差显微镜观察细胞的形态及萌出时间;Western Blot、免疫组织化学法观察HCECs的泵相关功能蛋白(zona occludens protein-1,ZO-1)及Na+-K+-ATP酶的表达.Giemsa染色细胞克隆实验、免疫组织化学及流式细胞学检测Ki67阳性率的方法比较HCECs的增殖能力;流式细胞学方法检查细胞周期及细胞凋亡情况.Western Blot和免疫组织化学方法检测细胞周期负性调节蛋白P21的水平,初步探讨其可能的作用机制.结果 原代培养时,25% ESC-CM组培养的HCECs P2细胞爬出,细胞形态呈典型多角形结构.CEM在P2时细胞形态变大,失去了多角形结构.25% ESC-CM组和CEM组均表达ZO-1、Na+-K+-ATP酶.25% ESC-CM组的Ki67阳性率、克隆形成数量、进入到细胞周期S期和G2期的比例均高于CEM组(均为P<0.05).25% ESC-CM组的细胞凋亡数量和P21阳性率均低于CEM组(均为P<0.05).结论 25% ESC-CM组可显著促进HCECs增殖;其作用可能是通过抑制P21蛋白的表达和抑制细胞凋亡实现的,为HCECs体外大量扩增提供了一种新方法.

  13. The Preliminary Experimental Study of Induced Differentiation of Embryonic Stem Cells into Corneal Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    Ling Yu; Jian Ge; Zhichong Wang; Bing Huang; Keming Yu; Chongde Long; Xigu Chen

    2001-01-01

    Purpose:To study preliminarily induced differentiation of embryonic stem cells intocorneal epithelial cells in vitro.Methods: Murine embryonic stem cells were co-cultured with Rabbit limbal cornealepithelial cells in Transwell system to induce differentiation. Mophological andimmunohistochemical examination were implemented.Results: The induced cells from embryonic stem cells have an epithelial appearance.The cells formed a network and were confluent into film gradually after beingco-cultured with rabbit limbal corneal epithelial cells for 24 ~ 96 hours. The cells rangedmosaic structure and localized together with clear rim. Most of the cells showedpolygonal appearance. Transmission electron microscope showed lots of microvilli on thesurface of induced cells and tight junctions between them. These epithelial-like cellsexpressed the corneal epithelial cell specific marker cytokeratin3/cytokeratinl2.Conclusion: The potential mechanism of the differentiation of murine embryonic stemcells into corneal epithelial cells induced by limbal corneal epithelial cell-derivedinducing activity is to be further verified.

  14. Uso de viscoelásticos na facoemulsificação em cães portadores de catarata: efeitos sobre a pressão intraocular, a morfologia das células endoteliais e a espessura corneana Use of viscoelastic substances for the phacoemulsification in dogs with cataract: effects on the intraocular pressure, morphology of endothelial cells, and corneal thickness

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    J.L.V. Chiurciu

    2010-06-01

    Full Text Available Avaliaram-se as células endoteliais, a espessura corneana e a pressão intraocular (PIO de cães portadores de catarata madura, empregando-se viscoelástico à base de hialuronato de sódio 3% e sulfato de condroitina 4% e hidroxipropilmetilcelulose 2%, utilizando-se 20 cães, distribuídos entre os dois grupos dos viscoelásticos. A técnica cirúrgica adotada foi a da facoemulsificação bimanual. As avaliações tonométricas foram efetuadas antes e após o ato cirúrgico, aos 1, 7, 14, 21, 28 e 60 dias de pós-operatório, e a microscopia especular, antes e após 7, 28 e 60 dias. Não houve diferença estatística entre os grupos quanto à PIO, com exceção aos 14 dias, em que se observou maior PIO com o uso de hialuronato de sódio 3% e sulfato de condroitina 4%. Não houve diferença entre os grupos quanto aos parâmetros relacionados ao endotélio, com diminuição discreta da densidade celular endotelial e aumento da área celular com a utilização de hidroxipropilmetilcelulose 2%. A utilização de ambos os dispositivos viscoelásticos analisados é recomendada para o procedimento de facoemulsificação em cães.The endothelial cells, the corneal thickness, and the intraocular pressure (IOP were evaluated in dogs with cataract, using viscoelastic substances based on 3% sodium hyaluronate and 4% chondroitin sulfate and comparing them with 2% hydroxypropylmethylcellulose. Twenty dogs were distributed in two groups of ten, each using one viscoelastic material. The surgical technique was bimanual phacoemulsification. The tonometric evaluations were made before and at one, seven, 14, 21, 28, and 60 days after the surgery and the specular microscopy before and after seven, 28, and 60 days. No statistical difference between groups was found according to IOP, except at 14 days, which was significantly higher with the use of 3% sodium hyaluronate and 4% chondroitin sulfate. There was no statistical difference between the groups considering

  15. Protective Effects of Trehalose on the Corneal Epithelial Cells

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    Pasquale Aragona

    2014-01-01

    Full Text Available Purpose. Aim of the present work was to evaluate the effects of the trehalose on the corneal epithelium undergoing alcohol delamination. Methods. Twelve patients undergoing laser subepithelial keratomileusis (LASEK were consecutively included in the study. The right eyes were pretreated with 3% trehalose eye drops, whilst left eyes were used as control. Epithelial specimens were processed for cells vitality assessment, apoptosis, and light and transmission electron microscopy; a morphometric analysis was performed in both groups. Results. In both trehalose-untreated eyes (TUE and trehalose-treated eyes (TTE, the percentage of vital cells was similar and no apoptotic cells were observed. In TUE, the corneal epithelium showed superficial cells with reduced microfolds, wing cells with vesicles and dilated intercellular spaces, and dark basal cells with vesicles and wide clefts. In TTE, superficial and wing cells were better preserved, and basal cells were generally clear with intracytoplasmatic vesicles. The morphometric analysis showed statistically significant differences between the two groups: the TTE epithelial height was higher, the basal cells showed larger area and clearer cytoplasm. The distribution of desmosomes and hemidesmosomes was significantly different between the groups. Conclusions. Trehalose administration better preserved morphological and morphometric features of alcohol-treated corneal epithelium, when compared to controls.

  16. Microvascular endothelial cells of the corpus luteum

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    Spanel-Borowski Katherina

    2003-11-01

    Full Text Available Abstract The cyclic nature of the capillary bed in the corpus luteum offers a unique experimental model to examine the life cycle of endothelial cells, involving discrete physiologically regulated steps of angiogenesis, blood vessel maturation and blood vessel regression. The granulosa cells and theca cells of the developing antral follicle and the steroidogenic cells of the corpus luteum produce and respond to angiogenic factors and vasoactive peptides. Following ovulation the neovascularization during the early stages of corpus luteum development has been compared to the rapid angiogenesis observed during tumor formation. On the other end of the spectrum, the microvascular endothelial cells are the first cells to undergo apoptosis at the onset of corpus luteum regression. Important insights on the morphology and function of luteal endothelial cells have been gained from a combination of in vitro and in vivo studies on endothelial cells. Endothelial cells communicate with cells comprising the functional unit of the corpus luteum, i.e., other vascular cells, steroidogenic cells, and immune cells. This review is designed to provide an overview of the types of endothelial cells present in the corpus luteum and their involvement in corpus luteum development and regression. Available evidence indicates that microvascular endothelial cells of the corpus luteum are not alike, and may differ during the process of angiogenesis and angioregression. The contributions of vasoactive peptides generated by the luteal endothelin-1 and the renin-angiotensin systems are discussed in context with the function of endothelial cells during corpus luteum formation and regression. The ability of two cytokines, tumor necrosis factor alpha and interferon gamma, are evaluated as paracrine mediators of endothelial cell function during angioregression. Finally, chemokines are discussed as a vital endothelial cell secretory products that contribute to the recruitment of

  17. Pseudophakodonesis and corneal endothelial contact: direct observations by high-speed cinematography.

    Science.gov (United States)

    Jacobs, P M; Cheng, H; Price, N C

    1983-10-01

    High-speed cinematography was used to observe the movement of Federov type I lens implants within the anterior chamber. Our measurements suggest that in most patients contact between the lens implant and corneal endothelium does not occur.

  18. Cell therapy of congenital corneal diseases with umbilical mesenchymal stem cells: lumican null mice.

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    Hongshan Liu

    Full Text Available BACKGROUND: Keratoplasty is the most effective treatment for corneal blindness, but suboptimal medical conditions and lack of qualified medical personnel and donated cornea often prevent the performance of corneal transplantation in developing countries. Our study aims to develop alternative treatment regimens for congenital corneal diseases of genetic mutation. METHODOLOGY/PRINCIPAL FINDINGS: Human mesenchymal stem cells isolated from neonatal umbilical cords were transplanted to treat thin and cloudy corneas of lumican null mice. Transplantation of umbilical mesenchymal stem cells significantly improved corneal transparency and increased stromal thickness of lumican null mice, but human umbilical hematopoietic stem cells failed to do the same. Further studies revealed that collagen lamellae were re-organized in corneal stroma of lumican null mice after mesenchymal stem cell transplantation. Transplanted umbilical mesenchymal stem cells survived in the mouse corneal stroma for more than 3 months with little or no graft rejection. In addition, these cells assumed a keratocyte phenotype, e.g., dendritic morphology, quiescence, expression of keratocyte unique keratan sulfated keratocan and lumican, and CD34. Moreover, umbilical mesenchymal stem cell transplantation improved host keratocyte functions, which was verified by enhanced expression of keratocan and aldehyde dehydrogenase class 3A1 in lumican null mice. CONCLUSIONS/SIGNIFICANCE: Umbilical mesenchymal stem cell transplantation is a promising treatment for congenital corneal diseases involving keratocyte dysfunction. Unlike donated corneas, umbilical mesenchymal stem cells are easily isolated, expanded, stored, and can be quickly recovered from liquid nitrogen when a patient is in urgent need.

  19. Progress in corneal wound healing.

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    Ljubimov, Alexander V; Saghizadeh, Mehrnoosh

    2015-11-01

    Corneal wound healing is a complex process involving cell death, migration, proliferation, differentiation, and extracellular matrix remodeling. Many similarities are observed in the healing processes of corneal epithelial, stromal and endothelial cells, as well as cell-specific differences. Corneal epithelial healing largely depends on limbal stem cells and remodeling of the basement membrane. During stromal healing, keratocytes get transformed to motile and contractile myofibroblasts largely due to activation of transforming growth factor-β (TGF-β) system. Endothelial cells heal mostly by migration and spreading, with cell proliferation playing a secondary role. In the last decade, many aspects of wound healing process in different parts of the cornea have been elucidated, and some new therapeutic approaches have emerged. The concept of limbal stem cells received rigorous experimental corroboration, with new markers uncovered and new treatment options including gene and microRNA therapy tested in experimental systems. Transplantation of limbal stem cell-enriched cultures for efficient re-epithelialization in stem cell deficiency and corneal injuries has become reality in clinical setting. Mediators and course of events during stromal healing have been detailed, and new treatment regimens including gene (decorin) and stem cell therapy for excessive healing have been designed. This is a very important advance given the popularity of various refractive surgeries entailing stromal wound healing. Successful surgical ways of replacing the diseased endothelium have been clinically tested, and new approaches to accelerate endothelial healing and suppress endothelial-mesenchymal transformation have been proposed including Rho kinase (ROCK) inhibitor eye drops and gene therapy to activate TGF-β inhibitor SMAD7. Promising new technologies with potential for corneal wound healing manipulation including microRNA, induced pluripotent stem cells to generate corneal

  20. Progress in corneal wound healing

    Science.gov (United States)

    Ljubimov, Alexander V.; Saghizadeh, Mehrnoosh

    2015-01-01

    Corneal wound healing is a complex process involving cell death, migration, proliferation, differentiation, and extracellular matrix remodeling. Many similarities are observed in the healing processes of corneal epithelial, stromal and endothelial cells, as well as cell-specific differences. Corneal epithelial healing largely depends on limbal stem cells and remodeling of the basement membrane. During stromal healing, keratocytes get transformed to motile and contractile myofibroblasts largely due to activation of transforming growth factor-β system. Endothelial cells heal mostly by migration and spreading, with cell proliferation playing a secondary role. In the last decade, many aspects of wound healing process in different parts of the cornea have been elucidated, and some new therapeutic approaches have emerged. The concept of limbal stem cells received rigorous experimental corroboration, with new markers uncovered and new treatment options including gene and microRNA therapy tested in experimental systems. Transplantation of limbal stem cell-enriched cultures for efficient re-epithelialization in stem cell deficiency and corneal injuries has become reality in clinical setting. Mediators and course of events during stromal healing have been detailed, and new treatment regimens including gene (decorin) and stem cell therapy for excessive healing have been designed. This is a very important advance given the popularity of various refractive surgeries entailing stromal wound healing. Successful surgical ways of replacing the diseased endothelium have been clinically tested, and new approaches to accelerate endothelial healing and suppress endothelial-mesenchymal transformation have been proposed including Rho kinase (ROCK) inhibitor eye drops and gene therapy to activate TGF-β inhibitor SMAD7. Promising new technologies with potential for corneal wound healing manipulation including microRNA, induced pluripotent stem cells to generate corneal epithelium, and

  1. Effects of N-acetylcysteine on matrix metalloproteinase-9 secretion and cell migration of human corneal epithelial cells

    OpenAIRE

    Ramaesh, T; Ramaesh, K; Riley, S C; West, J.D.; Dhillon, B

    2012-01-01

    Matrix metalloproteinase-9 (MMP-9) secreted by corneal epithelial cells has a role in the remodelling of extracellular matrix and migration of epithelial cells. Elevated levels of MMP-9 activity in the ocular surface may be involved in the pathogenesis of corneal diseases. N-acetylcysteine (NAC) has been used to treat corneal diseases, including recurrent epithelial erosions. In this study, its effects on the MMP-9 secretion and human corneal epithelial (HCE) cell migration were evaluated in ...

  2. Corneal blindness and xenotransplantation.

    Science.gov (United States)

    Lamm, Vladimir; Hara, Hidetaka; Mammen, Alex; Dhaliwal, Deepinder; Cooper, David K C

    2014-01-01

    Approximately 39 million people are blind worldwide, with an estimated 285 million visually impaired. The developing world shoulders 90% of the world's blindness, with 80% of causative diseases being preventable or treatable. Blindness has a major detrimental impact on the patient, community, and healthcare spending. Corneal diseases are significant causes of blindness, affecting at least 4 million people worldwide. The prevalence of corneal disease varies between parts of the world. Trachoma, for instance, is the second leading cause of blindness in Africa, after cataracts, but is rarely found today in developed nations. When preventive strategies have failed, corneal transplantation is the most effective treatment for advanced corneal disease. The major surgical techniques for corneal transplantation include penetrating keratoplasty (PK), anterior lamellar keratoplasty, and endothelial keratoplasty (EK). Indications for corneal transplantation vary between countries, with Fuchs' dystrophy being the leading indication in the USA and keratoconus in Australia. With the exception of the USA, where EK will soon overtake PK as the most common surgical procedure, PK is the overwhelming procedure of choice. Success using corneal grafts in developing nations, such as Nepal, demonstrates the feasibility of corneal transplantation on a global scale. The number of suitable corneas from deceased human donors that becomes available will never be sufficient, and so research into various alternatives, for example stem cells, amniotic membrane transplantation, synthetic and biosynthetic corneas, and xenotransplantation, is progressing. While each of these has potential, we suggest that xenotransplantation holds the greatest potential for a corneal replacement. With the increasing availability of genetically engineered pigs, pig corneas may alleviate the global shortage of corneas in the near future.

  3. Differentiation of embryonic stem cells into corneal epithelium

    Institute of Scientific and Technical Information of China (English)

    WANG Zhichong; LIU Jingbo; GE Jian; HUANG Bing; GAO Qianying; LIU Bingqian; WANG Linghua; YU Ling; FAN Zhigang; LU Xiaoming

    2005-01-01

    Our project was to determine whether embryonic stem (ES) cells could be induced to differentiate into corneal epithelia by superficial corneoscleral limbal stroma. To achieve this goal, ES-GFP cell line D3 was pre-induced by retinoic acid (RA). The pre-induced cells were seeded on deepithelialized superficial corneoscleral slices (SCSS) to form a monolayer, and divided into three groups. Group 1 was cultured and passaged in vitro for direct detection. Group 2 was exposed to air-liquid interfaces for 10 days and implanted into the subcutaneous layer of nude mice for 2 weeks for further induction in vivo. Group 3 was cultured in vitro without any inducing factors for control. There were no teratomas found in nude mice which were implanted with differentiated ES cells after two weeks. The differentiated cells showed an appearance of epithelia both in vitro and in vivo. Expression of CK3, P63 and PCNA was detected by immunohistochemical staining in the differentiated cells in group 1 and 2. Microvillis and zonula occludens were observed on the surface of the differentiated cells under an electron microscope. In the control group, ES cells differentiated freely without any inducing factors. Most cells were shed and formed a neuronal dendrite-like structure, and a minority of cells appeared polymorphic. These results demonstrate that ES cells can differentiate into corneal epithelia on the surface of SCSS under the controlled condition. Differentiated ES cells could be used as epithelial seeding cells for the reconstruction of ocular surface and corneal tissue engineering in the future.

  4. Study on effect of phacoemulsification combined with goniosynechialysis on corneal endothelial cells for the treatment of angle closure glaucoma%超声乳化联合前房角分离术对伴有白内障的闭角型青光眼角膜内皮细胞的影响

    Institute of Scientific and Technical Information of China (English)

    汪涛; 颜华

    2016-01-01

    AIM:To observe the effect of phacoemulsification and intraocular lens ( IOL ) implantation combined with goniosynechialysis on corneal endothelial cells for the treatment of primary angle closure glaucoma ( PACG ) combined cataract, and to analyze the relative factors.METHODS: Ninety-five eyes of 95 patients with PACG combined cataract were documented in this study. Twenty-two patients were male, and 73 were female. The age ranged from 46 to 85y old with a mean of(66±7) y. All patients were examined for endothelial cell count ( ECC ) , intraocular pressure ( IOP ) and best corrected visual acuity ( BCVA ) 1wk, 1, 2, 3 and 6mo after operation. Meanwhile, the range of anterior chamber closure and anterior chamber depth ( ACD ) were recorded before operation and postoperative 6mo. RESULTS: The mean IOP was 36. 1±4. 3mmHg ( 28-42mmHg) preoperatively and 15. 8±3. 5mmHg ( 8-28 mmHg)(1mmHg=0. 133kPa) 6mo after operation. There was a decreasing trend in IOP after operation( t=17. 173, P CONCLUSION: Phacoemulsification and IOL -implantation combined with goniosynechialysis is an effective method to treat PACG combined cataract. It can increase BCVA and decrease IOP. ECC decreases after operation, but it is in the normal range. It is a safe and effective operation mode.%目的:探讨伴有白内障的原发性闭角型青光眼( primary angle closure glaucoma ,PACG)采用超声乳化白内障吸出(phacoemulsification,Phaco)人工晶状体(IOL)植入联合前房角分离术治疗后角膜内皮细胞的变化,并分析相关影响因素。  方法:回顾分析接受Phaco+IOL联合前房角分离术治疗的PACG合并白内障患者95例95眼,其中女73例,男22例,年龄46~85(平均66±7)岁。记录并分析术前及术后1wk,1、2、3、6mo 最佳矫正视力( BCVA)、眼内压( IOP)及角膜内皮细胞计数( endothelial cell count,ECC),同时记录术前及术后6 mo 前房深度( anterior chamber depth,ACD)以及前房角关闭范围。随访8mo~2a。 

  5. Endothelial progenitor cells in cardiovascular diseases

    Institute of Scientific and Technical Information of China (English)

    Poay; Sian; Sabrina; Lee; Kian; Keong; Poh

    2014-01-01

    Endothelial dysfunction has been associated with the development of atherosclerosis and cardiovascular diseases. Adult endothelial progenitor cells(EPCs) are derived from hematopoietic stem cells and are capable of forming new blood vessels through a process of vas-culogenesis. There are studies which report correlations between circulating EPCs and cardiovascular risk fac-tors. There are also studies on how pharmacotherapies may influence levels of circulating EPCs. In this review, we discuss the potential role of endothelial progenitor cells as both diagnostic and prognostic biomarkers. In addition, we look at the interaction between cardio-vascular pharmacotherapies and endothelial progenitor cells. We also discuss how EPCs can be used directly and indirectly as a therapeutic agent. Finally, we evalu-ate the challenges facing EPC research and how these may be overcome.

  6. Differentiation and molecular profiling of human embryonic stem cell-derived corneal epithelial cells.

    Science.gov (United States)

    Brzeszczynska, J; Samuel, K; Greenhough, S; Ramaesh, K; Dhillon, B; Hay, D C; Ross, J A

    2014-06-01

    It has been suggested that the isolation of scalable populations of limbal stem cells may lead to radical changes in ocular therapy. In particular, the derivation and transplantation of corneal stem cells from these populations may result in therapies providing clinical normality of the diseased or damaged cornea. Although feasible in theory, the lack of donor material in sufficient quantity and quality currently limits such a strategy. A potential scalable source of corneal cells could be derived from pluripotent stem cells (PSCs). We developed an in vitro and serum-free corneal differentiation model which displays significant promise. Our stepwise differentiation model was designed with reference to development and gave rise to cells which displayed similarities to epithelial progenitor cells which can be specified to cells displaying a corneal epithelial phenotype. We believe our approach is novel, provides a robust model of human development and in the future, may facilitate the generation of corneal epithelial cells that are suitable for clinical use. Additionally, we demonstrate that following continued cell culture, stem cell-derived corneal epithelial cells undergo transdifferentiation and exhibit squamous metaplasia and therefore, also offer an in vitro model of disease.

  7. Generation of Corneal Keratocytes from Human Embryonic Stem Cells.

    Science.gov (United States)

    Hertsenberg, Andrew J; Funderburgh, James L

    2016-01-01

    Human Embryonic Stem Cells (hESC) offer an important resource as a limitless supply of any differentiated cell type of the human body. Keratocytes, cells from the corneal stroma, may have the potential for restoration of vision in cell therapy and biomedical engineering applications, but these specialized cells are not readily expanded in vitro. Here we describe a two-part method to produce keratocytes from the H1 hESC cell line. The hESC cells, maintained and expanded in feeder-free culture medium are first differentiated to neural crest cells using the stromal-derived inducing activity (SDIA) of the PA6 mouse embryonic fibroblast cell line. The resulting neural crest cells are selected by their expression of cell-surface CD271 and subsequently cultured as 3D pellets in a defined differentiation medium to induce a keratocyte phenotype.

  8. Effect of culture medium on propagation and phenotype of corneal stroma-derived stem cells.

    Science.gov (United States)

    Sidney, Laura E; Branch, Matthew J; Dua, Harminder S; Hopkinson, Andrew

    2015-12-01

    The limbal area of the corneal stroma has been identified as a source of mesenchymal-like stem cells, which have potential for exploitation as a cell therapy. However, the optimal culture conditions are disputed and few direct media comparisons have been performed. In this report, we evaluated several media types to identify the optimal for inducing an in vitro stem cell phenotype. Primary human corneal stroma-derived stem cells (CSSCs) were extracted from corneoscleral rims. Culture in seven different media types was compared: Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS); M199 with 20% FBS; DMEM-F12 with 20% serum replacement, basic fibroblast growth factor and leukemia inhibitory factor (SCM); endothelial growth medium (EGM); semi-solid MethoCult; serum-free keratinocyte medium (K-SFM); and StemPro-34. Effects on proliferation, morphology, protein and messenger RNA expression were evaluated. All media supported proliferation of CSSCs with the exception of K-SFM and StemPro-34. Morphology differed between media: DMEM produced large cells, whereas EGM produced very small cells. Culture in M199 produced a typical mesenchymal stromal cell phenotype with high expression of CD105, CD90 and CD73 but not CD34. Culture in SCM produced a phenotype more reminiscent of a progenitor cell type with expression of CD34, ABCG2, SSEA-4 and PAX6. Culture medium can significantly influence CSSC phenotype. SCM produced a cell phenotype closest to that of a pluripotent stem cell, and we consider it to be the most appropriate for development as a clinical-grade medium for the production of CSSC phenotypes suitable for cell therapy. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  9. Hyaluronate Acid-Dependent Protection and Enhanced Corneal Wound Healing against Oxidative Damage in Corneal Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Jing Zhong

    2016-01-01

    Full Text Available Purpose. To evaluate the effects and mechanism of exogenous hyaluronate (HA in promoting corneal wound healing. Methods. Human corneal epithelial cells (HCECs were incubated with different concentrations of HA to evaluate their efficiency in promoting cell migration and their modulation of repair factors. After inducing hyperosmolar conditions, the cell morphologies, cell apoptosis, and expression levels of TNF-α and MMP-9 were detected to assess the protective role of HA. Corneal epithelium-injured rat models were established to test the therapeutic effects of 0.3% HA. Then, the wound healing rates, the RNA expression levels of inflammatory cytokines, and repair factors were examined. Results. HCECs in the 0.03% and 0.3% HA groups showed fewer morphological alterations and lower rates of cell apoptosis following preincubation with HA under hyperosmolar conditions, as well as the expression levels of MMP-9 and TNF-α. In the rat model, the areas of fluorescein staining in the corneas of 0.3% HA group were significantly smaller than the control group. The expression levels of IL-1β and MMP-9 were decreased, while CD44 and FN were increased in the 0.3% HA group. Conclusion. HA enhanced corneal epithelial cell wound healing by promoting cell migration, upregulating repair responses, and suppressing inflammatory responses.

  10. Hyaluronate Acid-Dependent Protection and Enhanced Corneal Wound Healing against Oxidative Damage in Corneal Epithelial Cells.

    Science.gov (United States)

    Zhong, Jing; Deng, Yuqing; Tian, Bishan; Wang, Bowen; Sun, Yifang; Huang, Haixiang; Chen, Ling; Ling, Shiqi; Yuan, Jin

    2016-01-01

    Purpose. To evaluate the effects and mechanism of exogenous hyaluronate (HA) in promoting corneal wound healing. Methods. Human corneal epithelial cells (HCECs) were incubated with different concentrations of HA to evaluate their efficiency in promoting cell migration and their modulation of repair factors. After inducing hyperosmolar conditions, the cell morphologies, cell apoptosis, and expression levels of TNF-α and MMP-9 were detected to assess the protective role of HA. Corneal epithelium-injured rat models were established to test the therapeutic effects of 0.3% HA. Then, the wound healing rates, the RNA expression levels of inflammatory cytokines, and repair factors were examined. Results. HCECs in the 0.03% and 0.3% HA groups showed fewer morphological alterations and lower rates of cell apoptosis following preincubation with HA under hyperosmolar conditions, as well as the expression levels of MMP-9 and TNF-α. In the rat model, the areas of fluorescein staining in the corneas of 0.3% HA group were significantly smaller than the control group. The expression levels of IL-1β and MMP-9 were decreased, while CD44 and FN were increased in the 0.3% HA group. Conclusion. HA enhanced corneal epithelial cell wound healing by promoting cell migration, upregulating repair responses, and suppressing inflammatory responses.

  11. A novel closed cell culture device for fabrication of corneal epithelial cell sheets.

    Science.gov (United States)

    Nakajima, Ryota; Kobayashi, Toyoshige; Moriya, Noboru; Mizutani, Manabu; Kan, Kazutoshi; Nozaki, Takayuki; Saitoh, Kazuo; Yamato, Masayuki; Okano, Teruo; Takeda, Shizu

    2015-11-01

    Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets.

  12. Aloe vera extract activity on human corneal cells.

    Science.gov (United States)

    Woźniak, Anna; Paduch, Roman

    2012-02-01

    Ocular diseases are currently an important problem in modern societies. Patients suffer from various ophthalmologic ailments namely, conjunctivitis, dry eye, dacryocystitis or degenerative diseases. Therefore, there is a need to introduce new treatment methods, including medicinal plants usage. Aloe vera [Aloe barbadensis Miller (Liliaceae)] possesses wound-healing properties and shows immunomodulatory, anti-inflammatory or antioxidant activities. NR uptake, MTT, DPPH• reduction, Griess reaction, ELISA and rhodamine-phalloidin staining were used to test toxicity, antiproliferative activity, reactive oxygen species (ROS) reduction, nitric oxide (NO) and cytokine level, and distribution of F-actin in cells, respectively. The present study analyzes the effect of Aloe vera extracts obtained with different solvents on in vitro culture of human 10.014 pRSV-T corneal cells. We found no toxicity of ethanol, ethyl acetate and heptane extracts of Aloe vera on human corneal cells. No ROS reducing activity by heptane extract and trace action by ethanol (only at high concentration 125 µg/ml) extract of Aloe vera was observed. Only ethyl acetate extract expressed distinct free radical scavenging effect. Plant extracts decreased NO production by human corneal cells as compared to untreated controls. The cytokine (IL-1β, IL-6, TNF-α and IL-10) production decreased after the addition of Aloe vera extracts to the culture media. Aloe vera contains multiple pharmacologically active substances which are capable of modulating cellular phenotypes and functions. Aloe vera ethanol and ethyl acetate extracts may be used in eye drops to treat inflammations and other ailments of external parts of the eye such as the cornea.

  13. Endothelial cells derived from human embryonic stem cells

    Science.gov (United States)

    Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert

    2002-04-01

    Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  14. Reduced Ang2 expression in aging endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Hohensinner, P.J., E-mail: philipp.hohensinner@meduniwien.ac.at [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ebenbauer, B. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Kaun, C.; Maurer, G. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Huber, K. [Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); 3rd Medical Department, Wilhelminenhospital, Vienna (Austria); Sigmund Freud University, Medical Faculty, Vienna (Austria); Wojta, J. [Department of Internal Medicine II, Medical University of Vienna, Vienna (Austria); Ludwig Boltzmann Cluster for Cardiovascular Research, Vienna (Austria); Core Facilities, Medical University of Vienna, Vienna (Austria)

    2016-06-03

    Aging endothelial cells are characterized by increased cell size, reduced telomere length and increased expression of proinflammatory cytokines. In addition, we describe here that aging reduces the migratory distance of endothelial cells. Furthermore, we observe an increase of the quiescence protein Ang1 and a decrease of the endothelial activation protein Ang2 upon aging. Supplementing Ang2 to aged endothelial cells restored their migratory capacity. We conclude that aging shifts the balance of the Ang1/Ang2 network favouring a quiescent state. Activation of endothelial cells in aging might be necessary to enhance wound healing capacities. -- Highlights: •Endothelial cells display signs of aging before reaching proliferative senescence. •Aging endothelial cells express more angiopoietin 1 and less angiopoietin 2 than young endothelial cells. •Migratory capacity is reduced in aging endothelial cells.

  15. The Effects of Vitamin A Compounds on Hyaluronic Acid Released from Cultured Rabbit Corneal Epithelial Cells and Keratocytes

    National Research Council Canada - National Science Library

    TOSHIDA, Hiroshi; TABUCHI, Nobuhito; KOIKE, Daisuke; KOIDE, Misao; SUGIYAMA, Keikichi; NAKAYASU, Kiyoo; KANAI, Atsushi; MURAKAMI, Akira

    2012-01-01

    .... Hyaluronic acid is produced by corneal epithelial cells and keratocytes in the eye. We investigated whether rabbit corneal epithelial cells and keratocytes release hyaluronic acid after exposure to vitamin A compounds...

  16. Dry Eye and Corneal Langerhans Cells in Systemic Lupus Erythematosus

    Directory of Open Access Journals (Sweden)

    Miklós D. Resch

    2015-01-01

    Full Text Available Purpose. Investigation of dry eye and corneal Langerhans cells (LCs in systemic lupus erythematosus (SLE. Methods. Prospective consecutive case series of 27 SLE patients and 27 control subjects. Dry eye was evaluated by lid-parallel conjunctival folds (LIPCOF, Schirmer test, tear break-up time (TBUT, and ocular surface disease index (OSDI questionnaire. In vivo investigation of corneal LCs density and morphology (LCM was performed with confocal corneal microscopy (Heidelberg Retina Tomograph with Rostock Cornea Module. Results. Tear production and stability were pathological in SLE subjects compared to control (Schirmer: 8.45 ± 9.82 mm/5 min versus 11.67 ± 3.21 mm/5 min; TBUT: 6.86 ± 3.53 s versus 11.09 ± 3.37 s. OSDI was significantly greater in SLE patients (25.95 ± 17.92 than in controls (11.06 ± 7.18. Central LC density was greater in SLE patients (43.08 ± 48.67 cell/mm2 than in controls (20.57 ± 21.04 cell/mm2. There was no difference in the peripheral LC density (124.78 ± 165.39 versus 78.00 ± 39.51 cell/mm2. LCM was higher in SLE patients in the centre (1.43 ± 0.79 and in the periphery (2.89 ± 0.42 compared to controls (centre: 1.00 ± 0.69, periphery: 2.35 ± 0.54. Conclusions. Significant changes in dry eye parameters and marked increase of central LCs could be demonstrated in SLE patients. SLE alters not only the LC density but also the morphology, modifies corneal homeostasis, and might contribute to the development of dry eye.

  17. Utilização do programa Cells Analyser® no estudo comparativo entre imagens do endotélio de córneas obtidas por microscopia especular Using of Cells Analyser® software in the study of image of corneal specular microscope endothelial samples

    Directory of Open Access Journals (Sweden)

    Cinthia Mendonça de Melo

    2008-02-01

    Full Text Available OBJETIVOS: Conhecer diferenças entre o número de células, área endotelial avaliada e erro amostral, comparando amostras endoteliais de uma única imagem endotelial, com amostras endoteliais constituídas pelo número de células e imagens, orientadas por software específico. MÉTODOS: Foi realizado estudo transversal, comparando as amostras endoteliais com e sem a intervenção do software. A amostra foi composta de 157 olhos. Foram criados 2 grupos: Grupo 1: composto pelos dados amostrais da primeira imagem dos exames; Grupo 2: composto pelos dados amostrais que consideram o número de imagens necessárias para que o exame seja considerado completo pelo software Cells Analyzer PAT. REC. (Grau de confiança 95% e erro relativo 0,05. Os dados amostrais comparados foram número de células contadas, área da amostra endotelial e erro amostral. Utilizou-se o teste t Student bicaudal, para amostras pareadas, com nível de 99% (pPURPOSE: To describe differences between number of cells, evaluated endothelial area and relative error comparing endothelial samples from one endothelial image with endothelial samples obtained by guidance of a specific software, with number of cells and images as many as necessary. METHODS: A transversal study was performed, comparing the endothelial samples with and without intervention of the software. The sample consisted of 157 eyes. They were divided into 2 groups. Group 1 consisted of data of the first image and group 2 consisted of data from as many images as necessary for a complete examination guided by the Cells Analyser software (95% confidence interval and 0.05 relative error. The evaluated data were number of counted cells, equivalent evaluated field (mm² and relative error. Student's t test with 99% confidence interval (p<0.01 was used. RESULTS: The compared data showed statistically significant differences between groups 1 and 2 in cell count (p=4x10-24, evaluated endothelial field (p=2x10-18 and

  18. [Protection of corneal endothelium from apoptosis by gene and cell therapy].

    Science.gov (United States)

    Fuchsluger, T A

    2016-06-01

    Protection of corneal endothelium from apoptosis using gene and cell therapy is in a translational phase. This approach offers advantages for eye banking and after transplantation. Safe vehicles for gene or cell therapeutic transduction of corneal endothelium with nucleic acids are available. This strategy will be further developed in consultation with the Paul Ehrlich Institute and European regulatory authorities.

  19. A comparative evaluation of corneal epithelial cell cultures for assessing ocular permeability.

    Science.gov (United States)

    Becker, Ulrich; Ehrhardt, Carsten; Schneider, Marc; Muys, Leon; Gross, Dorothea; Eschmann, Klaus; Schaefer, Ulrich F; Lehr, Claus-Michael

    2008-02-01

    The purpose of this study was to evaluate the potential value of different epithelial cell culture systems as in vitro models for studying corneal permeability. Transformed human corneal epithelial (HCE-T) cells and Statens Serum Institut rabbit corneal (SIRC) cells were cultured on permeable filters. SkinEthic human corneal epithelium (S-HCE) and Clonetics human corneal epithelium (C-HCE) were received as ready-to-use systems. Excised rabbit corneas (ERCs) and human corneas (EHCs) were mounted in Ussing chambers, and used as references. Barrier properties were assessed by measuring transepithelial electrical resistance, and by determining the apparent permeability of markers with different physico-chemical properties, namely, fluorescein, sodium salt; propranolol hydrochloride; moxaverine hydrochloride; timolol hydrogenmaleate; and rhodamine 123. SIRC cells and the S-HCE failed to develop epithelial barrier properties, and hence were unable to distinguish between the permeation markers. Barrier function and the power to differentiate compound permeabilities were evident with HCE-T cells, and were even more pronounced in the case of C-HCE, corresponding very well with data from ERCs and EHCs. A net secretion of rhodamine 123 was not observed with any of the models, suggesting that P-glycoprotein or similar efflux systems have no significant effects on corneal permeability. Currently available corneal epithelial cell culture systems show differences in epithelial barrier function. Systems lacking functional cell-cell contacts are of limited value for assessing corneal permeability, and should be critically evaluated for other purposes.

  20. Microvesicles Derived from Indoxyl Sulfate Treated Endothelial Cells Induce Endothelial Progenitor Cells Dysfunction.

    Science.gov (United States)

    Carmona, Andres; Guerrero, Fatima; Buendia, Paula; Obrero, Teresa; Aljama, Pedro; Carracedo, Julia

    2017-01-01

    Cardiovascular disease is a major cause of mortality in chronic kidney disease patients. Indoxyl sulfate (IS) is a typical protein-bound uremic toxin that cannot be effectively cleared by conventional dialysis. Increased IS is associated with the progression of chronic kidney disease and development of cardiovascular disease. After endothelial activation by IS, cells release endothelial microvesicles (EMV) that can induce endothelial dysfunction. We developed an in vitro model of endothelial damage mediated by IS to evaluate the functional effect of EMV on the endothelial repair process developed by endothelial progenitor cells (EPCs). EMV derived from IS-treated endothelial cells were isolated by ultracentrifugation and characterized for miRNAs content. The effects of EMV on healthy EPCs in culture were studied. We observed that IS activates endothelial cells and the generated microvesicles (IsEMV) can modulate the classic endothelial roles of progenitor cells as colony forming units and form new vessels in vitro. Moreover, 23 miRNAs were contained in IsEMV including four (miR-181a-5p, miR-4454, miR-150-5p, and hsa-let-7i-5p) that were upregulated in IsEMV compared with control endothelial microvesicles. Other authors have found that miR-181a-5p, miR-4454, and miR-150-5p are involved in promoting inflammation, apoptosis, and cellular senescence. Interestingly, we observed an increase in NFκB and p53, and a decrease in IκBα in EPCs treated with IsEMV. Our data suggest that IS is capable of inducing endothelial vesiculation with different membrane characteristics, miRNAs and other molecules, which makes maintaining of vascular homeostasis of EPCs not fully functional. These specific characteristics of EMV could be used as novel biomarkers for diagnosis and prognosis of vascular disease.

  1. Endothelial cells and the IGF system.

    Science.gov (United States)

    Bach, Leon A

    2015-02-01

    Endothelial cells line blood vessels and modulate vascular tone, thrombosis, inflammatory responses and new vessel formation. They are implicated in many disease processes including atherosclerosis and cancer. IGFs play a significant role in the physiology of endothelial cells by promoting migration, tube formation and production of the vasodilator nitric oxide. These actions are mediated by the IGF1 and IGF2/mannose 6-phosphate receptors and are modulated by a family of high-affinity IGF binding proteins. IGFs also increase the number and function of endothelial progenitor cells, which may contribute to protection from atherosclerosis. IGFs promote angiogenesis, and dysregulation of the IGF system may contribute to this process in cancer and eye diseases including retinopathy of prematurity and diabetic retinopathy. In some situations, IGF deficiency appears to contribute to endothelial dysfunction, whereas IGF may be deleterious in others. These differences may be due to tissue-specific endothelial cell phenotypes or IGFs having distinct roles in different phases of vascular disease. Further studies are therefore required to delineate the therapeutic potential of IGF system modulation in pathogenic processes. © 2015 Society for Endocrinology.

  2. MMSC-LIKE LIMBAL CELLS COTRANSPLANTATION PROMOTES LOCAL IMMUNOCORRECTION AND CORNEAL GRAFT TRANSPARENT RETENTION IN HIGH RISK KERATOPLASTY

    Directory of Open Access Journals (Sweden)

    S. A. Borzenok

    2014-01-01

    Full Text Available Aim was to evaluate clinical results of donor corneal graft survival in high-risk recipients in co-transplantation of preserved allogenic limbal grafts. Materials and methods. Two types of penetrative keratoplasties were carried out in patients with corneal graft opacities and high risk of rejection (n = 69. Co-transplantation of donor cornea and allogenic MMSC-like limbal cells in the form of limbal transplants was carried out in the 1st group (n = 36; in the 2nd group (n = 33 only the cornea was transplanted. Results. Observation of the patients during one year after surgery showed that the rate of transparent cornea engraftment increased in the 1st group (86,1 against 69,7% in the 2nd group. The density of endothelial cells was also higher in the 1st group (85,9 against 76,2% in the 2nd group. At the same time, progressive decreasing of pro-inflammatory cytokines (IL-6, IFNγ, TNFα and increasing of anti-inflammatory cytokines (IL-10, IL-1RA, TGFβ along with higher level of HLA-G5 were revealed in the recipients’ tear fluid in the 1st group in comparison to the 2nd group. Conclusion. Simultaneous transplantation of preserved limbal grafts with corneal graft in high-risk keratoplasty favors the transparent cornea engraftment, obviously, this is due to immunoregulatory activity of the MMSC-like limbal cells

  3. Phage display against corneal epithelial cells produced bioactive peptides that inhibit Aspergillus adhesion to the corneas.

    Directory of Open Access Journals (Sweden)

    Ge Zhao

    Full Text Available Dissection of host-pathogen interactions is important for both understanding the pathogenesis of infectious diseases and developing therapeutics for the infectious diseases like various infectious keratitis. To enhance the knowledge about pathogenesis infectious keratitis, a random 12-mer peptide phage display library was screened against cultured human corneal epithelial cells (HCEC. Fourteen sequences were obtained and BLASTp analysis showed that most of their homologue counterparts in GenBank were for defined or putative proteins in various pathogens. Based on known or predicted functions of the homologue proteins, ten synthetic peptides (Pc-A to Pc-J were measured for their affinity to bind cells and their potential efficacy to interfere with pathogen adhesion to the cells. Besides binding to HCEC, most of them also bound to human corneal stromal cells and umbilical endothelial cells to different extents. When added to HCEC culture, the peptides induced expression of MyD88 and IL-17 in HCEC, and the stimulated cell culture medium showed fungicidal potency to various extents. While peptides Pc-C and Pc-E inhibited Aspergillus fumigatus (A.f adhesion to HCEC in a dose-dependent manner, the similar inhibition ability of peptides Pc-A and Pc-B required presence of their homologue ligand Alb1p on A.f. When utilized in an eyeball organ culture model and an in vivo A.f keratitis model established in mouse, Pc-C and Pc-E inhibited fungal adhesion to corneas, hence decreased corneal disruption caused by inflammatory infiltration. Affinity pull-down of HCEC membrane proteins with peptide Pc-C revealed several molecules as potential receptors for this peptide. In conclusion, besides proving that phage display-selected peptides could be utilized to interfere with adhesion of pathogens to host cells, hence could be exploited for managing infectious diseases including infectious keratitis, we also proposed that the phage display technique and the

  4. Relationship between endothelial cell loss and microcoaxial phacoemulsification parameters in noncomplicated cataract surgery.

    Science.gov (United States)

    Mahdy, Mohamed Ae Soliman; Eid, Mohamed Z; Mohammed, Mahmoud Abdel-Badei; Hafez, Amr; Bhatia, Jagdish

    2012-01-01

    To assess the relationship between postoperative endothelial cell loss and microcoaxial phaco parameters using Ozil IP (Alcon Laboratories, Inc, Fort Worth, TX) in noncomplicated cataract surgery. In this prospective observational study, 120 consecutive cases of cataract patients with different grades of nuclear hardness underwent microcoaxial phacoemulsification through a 2.2-mm clear corneal incision. An Alcon Infinity Vision System with Ozil IP (Alcon Laboratories) was used with an Ozil torsional handpiece and a Kelman-style 45° phacoemulsification tip. Patients underwent preoperative and postoperative central endothelial cell counts. The study included 120 cases of age-related cataract whose mean age (standard deviation [SD]) was 59.68 years (9.47). There was a highly statistically significant endothelial cell loss (P phaco parameters. The Spearman's rank-order correlation coefficient values, rho, (ρ) were as follows: CDE (ρ = 0.425), aspiration time (ρ = 0.176), and volume (ρ = 0.278). Also, ECLoss% was significantly correlated with the grade of nuclear opalescence (Kendall's tau τ = 0.42). Microcoaxial phacoemulsification was efficient in removing noncomplicated cataracts; however a statistically significant endothelial cell loss was noted, especially with increased nuclear hardness. This endothelial cell loss was mostly related to the increased cumulative dissipated energy (CDE), aspiration time, and volume of balanced salt solution used.

  5. Rho GTPases and regulation of cell migration and polarization in human corneal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Aihua Hou

    Full Text Available PURPOSE: Epithelial cell migration is required for regeneration of tissues and can be defective in a number of ocular surface diseases. This study aimed to determine the expression pattern of Rho family small G-proteins in human corneal epithelial cells to test their requirement in directional cell migration. METHODS: Rho family small G-protein expression was assessed by reverse transcription-polymerase chain reaction. Dominant-inhibitory constructs encoding Rho proteins or Rho protein targeting small interfering RNA were transfected into human corneal epithelial large T antigen cells, and wound closure rate were evaluated by scratch wounding assay, and a complementary non-traumatic cell migration assay. Immunofluorescence staining was performed to study cell polarization and to assess Cdc42 downstream effector. RESULTS: Cdc42, Chp, Rac1, RhoA, TC10 and TCL were expressed in human corneal epithelial cells. Among them, Cdc42 and TCL were found to significantly affect cell migration in monolayer scratch assays. These results were confirmed through the use of validated siRNAs directed to Cdc42 and TCL. Scramble siRNA transfected cells had high percentage of polarized cells than Cdc42 or TCL siRNA transfected cells at the wound edge. We showed that the Cdc42-specific effector p21-activated kinase 4 localized predominantly to cell-cell junctions in cell monolayers, but failed to translocate to the leading edge in Cdc42 siRNA transfected cells after monolayer wounding. CONCLUSION: Rho proteins expressed in cultured human corneal epithelial cells, and Cdc42, TCL facilitate two-dimensional cell migration in-vitro. Although silencing of Cdc42 and TCL did not noticeably affect the appearance of cell adhesions at the leading edge, the slower migration of these cells indicates both GTP-binding proteins play important roles in promoting cell movement of human corneal epithelial cells.

  6. Endothelial cell seeding on crosslinked collagen : Effects of crosslinking on endothelial cell proliferation and functional parameters

    NARCIS (Netherlands)

    Wissink, MJB; van Luyn, MJA; Dijk, F; Poot, AA; Engbers, GHM; Beugeling, T; van Aken, WG; Feijen, J

    Endothelial cell seeding, a promising method to improve the performance of small-diameter vascular grafts, requires a suitable substrate, such as crosslinked collagen. Commonly used crosslinking agents such as glutaraldehyde and formaldehyde cause, however, cytotoxic reactions and thereby hamper

  7. System for tracking transplanted limbal epithelial stem cells in the treatment of corneal stem cell deficiency

    Science.gov (United States)

    Boadi, J.; Sangwal, V.; MacNeil, S.; Matcher, S. J.

    2015-03-01

    The prevailing hypothesis for the existence and healing of the avascular corneal epithelium is that this layer of cells is continually produced by stem cells in the limbus and transported onto the cornea to mature into corneal epithelium. Limbal Stem Cell Deficiency (LSCD), in which the stem cell population is depleted, can lead to blindness. LSCD can be caused by chemical and thermal burns to the eye. A popular treatment, especially in emerging economies such as India, is the transplantation of limbal stem cells onto damaged limbus with hope of repopulating the region. Hence regenerating the corneal epithelium. In order to gain insights into the success rates of this treatment, new imaging technologies are needed in order to track the transplanted cells. Optical Coherence Tomography (OCT) is well known for its high resolution in vivo images of the retina. A custom OCT system has been built to image the corneal surface, to investigate the fate of transplanted limbal stem cells. We evaluate two methods to label and track transplanted cells: melanin labelling and magneto-labelling. To evaluate melanin labelling, stem cells are loaded with melanin and then transplanted onto a rabbit cornea denuded of its epithelium. The melanin displays strongly enhanced backscatter relative to normal cells. To evaluate magneto-labelling the stem cells are loaded with magnetic nanoparticles (20-30nm in size) and then imaged with a custom-built, magneto-motive OCT system.

  8. Polysaccharide hydrogel combined with mesenchymal stem cells promotes the healing of corneal alkali burn in rats.

    Directory of Open Access Journals (Sweden)

    Yifeng Ke

    Full Text Available Corneal chemical burns are common ophthalmic injuries that may result in permanent visual impairment. Although significant advances have been achieved on the treatment of such cases, the structural and functional restoration of a chemical burn-injured cornea remains challenging. The applications of polysaccharide hydrogel and subconjunctival injection of mesenchymal stem cells (MSCs have been reported to promote the healing of corneal wounds. In this study, polysaccharide was extracted from Hardy Orchid and mesenchymal stem cells (MSCs were derived from Sprague-Dawley rats. Supplementation of the polysaccharide significantly enhanced the migration rate of primarily cultured rat corneal epithelial cells. We examined the therapeutic effects of polysaccharide in conjunction with MSCs application on the healing of corneal alkali burns in rats. Compared with either treatment alone, the combination strategy resulted in significantly better recovery of corneal epithelium and reduction in inflammation, neovascularization and opacity of healed cornea. Polysaccharide and MSCs acted additively to increase the expression of anti-inflammatory cytokine (TGF-β, antiangiogenic cytokine (TSP-1 and decrease those promoting inflammation (TNF-α, chemotaxis (MIP-1α and MCP-1 and angiogenesis (VEGF and MMP-2. This study provided evidence that Hardy Orchid derived polysaccharide and MSCs are safe and effective treatments for corneal alkali burns and that their benefits are additive when used in combination. We concluded that combination therapy with polysaccharide and MSCs is a promising clinical treatment for corneal alkali burns and may be applicable for other types of corneal disorder.

  9. Endothelial cell promotion of early liver and pancreas development.

    Science.gov (United States)

    Freedman, Deborah A; Kashima, Yasushige; Zaret, Kenneth S

    2007-01-01

    Different steps of embryonic pancreas and liver development require inductive signals from endothelial cells. During liver development, interactions between newly specified hepatic endoderm cells and nascent endothelial cells are crucial for the endoderm's subsequent growth and morphogenesis into a liver bud. Reconstitution of endothelial cell stimulation of hepatic cell growth with embryonic tissue explants demonstrated that endothelial signalling occurs independent of the blood supply. During pancreas development, midgut endoderm interactions with aortic endothelial cells induce Ptf1a, a crucial pancreatic determinant. Endothelial cells also have a later effect on pancreas development, by promoting survival of the dorsal mesenchyme, which in turn produces factors supporting pancreatic endoderm. A major goal of our laboratory is to determine the endothelial-derived molecules involved in these inductive events. Our data show that cultured endothelial cells induce Ptf1a in dorsal endoderm explants lacking an endogenous vasculature. We are purifying endothelial cell line product(s) responsible for this effect. We are also identifying endothelial-responsive regulatory elements in genes such as Ptf1a by genetic mapping and chromatin-based assays. These latter approaches will allow us to track endothelial-responsive signal pathways from DNA targets within progenitor cells. The diversity of organogenic steps dependent upon endothelial cell signalling suggests that cross-regulation of tissue development with its vasculature is a general phenomenon.

  10. Endothelial progenitor cell biology in ankylosing spondylitis.

    Science.gov (United States)

    Verma, Inderjeet; Syngle, Ashit; Krishan, Pawan

    2015-03-01

    Endothelial progenitor cells (EPCs) are unique populations which have reparative potential in overcoming endothelial damage and reducing cardiovascular risk. Patients with ankylosing spondylitis (AS) have increased risk of cardiovascular morbidity and mortality. The aim of this study was to investigate the endothelial progenitor cell population in AS patients and its potential relationships with disease variables. Endothelial progenitor cells were measured in peripheral blood samples from 20 AS and 20 healthy controls by flow cytometry on the basis of CD34 and CD133 expression. Disease activity was evaluated by using Bath Ankylosing Spondylitis Disease Activity Index (BASDAI). Functional ability was monitored by using Bath Ankylosing Spondylitis Functional Index (BASFI). EPCs were depleted in AS patients as compared to healthy controls (CD34(+) /CD133(+) : 0.027 ± 0.010% vs. 0.044 ± 0.011%, P < 0.001). EPC depletions were significantly associated with disease duration (r = -0.52, P = 0.01), BASDAI (r = -0.45, P = 0.04) and C-reactive protein (r = -0.5, P = 0.01). This is the first study to demonstrate endothelial progenitor cell depletion in AS patients. EPC depletions inversely correlate with disease duration, disease activity and inflammation, suggesting the pivotal role of inflammation in depletion of EPCs. EPC would possibly also serve as a therapeutic target for preventing cardiovascular disease in AS. © 2014 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd.

  11. Use of Corneal Confocal Microscopy to Detect Corneal Nerve Loss and Increased Dendritic Cells in Patients With Multiple Sclerosis.

    Science.gov (United States)

    Bitirgen, Gulfidan; Akpinar, Zehra; Malik, Rayaz A; Ozkagnici, Ahmet

    2017-07-01

    Multiple sclerosis (MS) is characterized by demyelination, axonal degeneration, and inflammation. Corneal confocal microscopy has been used to identify axonal degeneration in several peripheral neuropathies. To assess corneal subbasal nerve plexus morphologic features, corneal dendritic cell (DC) density, and peripapillary retinal nerve fiber layer (RNFL) thickness in patients with MS. This single-center, cross-sectional comparative study was conducted at a tertiary referral university hospital between May 27, 2016, and January 30, 2017. Fifty-seven consecutive patients with relapsing-remitting MS and 30 healthy, age-matched control participants were enrolled in the study. Corneal subbasal nerve plexus measures and DC density were quantified in images acquired with the laser scanning in vivo corneal confocal microscope, and peripapillary RNFL thickness was measured with spectral-domain optical coherence tomography. Corneal nerve fiber density, nerve branch density, nerve fiber length, DC density, peripapillary RNFL thickness, and association with the severity of neurologic disability as assessed by the Kurtzke Expanded Disability Status Scale (score range, 0-10; higher scores indicate greater disability) and Multiple Sclerosis Severity Score (score range, 0.01-9.99; higher scores indicate greater severity). Of the 57 participants with MS, 42 (74%) were female and the mean (SD) age was 35.4 (8.9) years; of the 30 healthy controls, 19 (63%) were female and the mean (SD) age was 34.8 (10.2) years. Corneal nerve fiber density (mean [SE] difference, -6.78 [2.14] fibers/mm2; 95% CI, -11.04 to -2.52; P = .002), nerve branch density (mean [SE] difference, -17.94 [5.45] branches/mm2; 95% CI, -28.77 to -7.10; P = .001), nerve fiber length (mean [SE] difference, -3.03 [0.89] mm/mm2; 95% CI, -4.81 to -1.25; P = .001), and the mean peripapillary RNFL thickness (mean [SE] difference, -17.06 [3.14] μm; 95% CI, -23.29 to -10.82; P < .001) were reduced in patients with MS compared

  12. Isolation, Characterization, and Transplantation of Cardiac Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Busadee Pratumvinit

    2013-01-01

    due to difficulties in isolation, cell heterogeneity, lack of specific markers to identify myocardial endothelial cells, and inadequate conditions to maintain long-term cultures. Herein, we developed a method for isolation, characterization, and expansion of cardiac endothelial cells applicable to study endothelial cell biology and clinical applications such as neoangiogenesis. First, we dissociated the cells from murine heart by mechanical disaggregation and enzymatic digestion. Then, we used flow cytometry coupled with specific markers to isolate endothelial cells from murine hearts. CD45+ cells were gated out to eliminate the hematopoietic cells. CD31+/Sca-1+ cells were isolated as endothelial cells. Cells isolated from atrium grew faster than those from ventricle. Cardiac endothelial cells maintain endothelial cell function such as vascular tube formation and acetylated-LDL uptake in vitro. Finally, cardiac endothelial cells formed microvessels in dorsal matrigel plug and engrafted in cardiac microvessels following intravenous and intra-arterial injections. In conclusion, our multicolor flow cytometry method is an effective method to analyze and purify endothelial cells from murine heart, which in turn can be ex vivo expanded to study the biology of endothelial cells or for clinical applications such as therapeutic angiogenesis.

  13. Human pluripotent stem cell-derived limbal epithelial stem cells on bioengineered matrices for corneal reconstruction.

    Science.gov (United States)

    Mikhailova, Alexandra; Ilmarinen, Tanja; Ratnayake, Anjula; Petrovski, Goran; Uusitalo, Hannu; Skottman, Heli; Rafat, Mehrdad

    2016-05-01

    Corneal epithelium is renewed by limbal epithelial stem cells (LESCs), a type of tissue-specific stem cells located in the limbal palisades of Vogt at the corneo-scleral junction. Acute trauma or inflammatory disorders of the ocular surface can destroy these stem cells, leading to limbal stem cell deficiency (LSCD) - a painful and vision-threatening condition. Treating these disorders is often challenging and complex, especially in bilateral cases with extensive damage. Human pluripotent stem cells (hPSCs) provide new opportunities for corneal reconstruction using cell-based therapy. Here, we investigated the use of hPSC-derived LESC-like cells on bioengineered collagen matrices in serum-free conditions, aiming for clinical applications to reconstruct the corneal epithelium and partially replace the damaged stroma. Differentiation of hPSCs towards LESC-like cells was directed using small-molecule induction followed by maturation in corneal epithelium culture medium. After four to five weeks of culture, differentiated cells were seeded onto bioengineered matrices fabricated as transparent membranes of uniform thickness, using medical-grade porcine collagen type I and a hybrid cross-linking technology. The bioengineered matrices were fully transparent, with high water content and swelling capacity, and parallel lamellar microstructure. Cell proliferation of hPSC-LESCs was significantly higher on bioengineered matrices than on collagen-coated control wells after two weeks of culture, and LESC markers p63 and cytokeratin 15, along with proliferation marker Ki67 were expressed even after 30 days in culture. Overall, hPSC-LESCs retained their capacity to self-renew and proliferate, but were also able to terminally differentiate upon stimulation, as suggested by protein expression of cytokeratins 3 and 12. We propose the use of bioengineered collagen matrices as carriers for the clinically-relevant hPSC-derived LESC-like cells, as a novel tissue engineering approach for

  14. Mycobacteria entry and trafficking into endothelial cells.

    Science.gov (United States)

    Baltierra-Uribe, Shantal Lizbeth; García-Vásquez, Manuel de Jesús; Castrejón-Jiménez, Nayeli Shantal; Estrella-Piñón, Mayra Patricia; Luna-Herrera, Julieta; García-Pérez, Blanca Estela

    2014-09-01

    Endothelial cells are susceptible to infection by mycobacteria, but the endocytic mechanisms that mycobacteria exploit to enter host cells and their mechanisms of intracellular transport are completely unknown. Using pharmacological inhibitors, we determined that the internalization of Mycobacterium tuberculosis (MTB), Mycobacterium smegmatis (MSM), and Mycobacterium abscessus (MAB) is dependent on the cytoskeleton and is differentially inhibited by cytochalasin D, nocodazole, cycloheximide, wortmannin, and amiloride. Using confocal microscopy, we investigated their endosomal trafficking by analyzing Rab5, Rab7, LAMP-1, and cathepsin D. Our results suggest that MSM exploits macropinocytosis to enter endothelial cells and that the vacuoles containing these bacteria fuse with lysosomes. Conversely, the entry of MTB seems to depend on more than one endocytic route, and the observation that only a subset of the intracellular bacilli was associated with phagolysosomes suggests that these bacteria are able to inhibit endosomal maturation to persist intracellularly. The route of entry for MAB depends mainly on microtubules, which suggests that MAB uses a different trafficking pathway. However, MAB is also able to inhibit endosomal maturation and can replicate intracellularly. Together, these findings provide the first evidence that mycobacteria modulate proteins of host endothelial cells to enter and persist within these cells.

  15. Proteomics of Fuchs' Endothelial Corneal Dystrophy support that the extracellular matrix of Descemet's membrane is disordered

    DEFF Research Database (Denmark)

    Poulsen, Ebbe Toftgaard; Dyrlund, Thomas F; Runager, Kasper;

    2014-01-01

    the protein profiles of diseased and control tissues using two relative quantitation MS methods. The first quantitation method based on the areas of the extracted ion chromatograms, quantified the 51 and 48 most abundant proteins of the Descemet's membrane/endothelial layer in patient and control tissues......, respectively, of which 10 were significantly regulated. The results indicated that the level of type VIII collagen was unaltered even though the protein previously has been implicated in familial early onset forms of the disease. Using the second relative quantitation method iTRAQ we identified 22...

  16. Transition of mesenchymal stem/stromal cells to endothelial cells

    NARCIS (Netherlands)

    M. Crisan (Mihaela)

    2013-01-01

    textabstractMesenchymal stem/stromal cells (MSCs) are heterogeneous. A fraction of these cells constitute multipotent cells that can self-renew and mainly give rise to mesodermal lineage cells such as adipocytes, osteocytes and chondrocytes. The ability of MSCs to differentiate into endothelial cell

  17. Comparison of Endothelial Cell Loss by Specular Microscopy ...

    African Journals Online (AJOL)

    ... was no clinically or statistically significant difference in endothelial cell loss or visual acuity between phacoemulsification and manual SICS at ... captured image was then transferred to the computer ... and iridocorneal endothelial syndrome.

  18. Effects of vascular endothelial growth factor on angiogenesis of the endothelial cells isolated from cavernous malformations

    Institute of Scientific and Technical Information of China (English)

    TAN YuZhen; ZHAO Yao; WANG HaiJie; ZHOU LiangFu; MAO Ying; LIU Rui; SHU Jia; WANG YongFei

    2008-01-01

    Human cerebral cavernous malformation (CM) is a common vascular malformation of the central nervous system. We have investigated the biological characteristics of CM endothelial cells and the cellular and molecular mechanisms of CM angiogenesis to offer new insights into exploring effective measures for treatment of this disease. The endothelial cells were isolated from CM tissue masses dissected during operation and expanded in vitro. Expression of VEGFR-1 and VEGFR-2 was examined with immunocytochemical staining. Proliferation, migration and tube formation of CM endothelial cells were determined using MTT, wounding and transmigration assays, and three-dimensional collagen type Ⅰ gel respectively. The endothelial cells were successfully isolated from the tissue specimens of 25 CMs dissected without dipolar electrocoagulation. The cells show the general characteristics of the vascular endothelial cells. Expression of VEGFR-1 and VEGFR-2 on the cells is higher than that on the normal cerebral microvascular endothelial cells. After treatment with VEGF, numbers of the proliferated and migrated cells, the maximal distance of cell migration and the length and area of capillary-like struc-tures formed in the three-dimensional collagen gel increase significantly. These results demonstrate that expression of VEGFR-1 and VEGFR-2 on CM endothelial cells is up-regulated. By binding to re-ceptors, VEGF may activate the downstream signaling pathways and promote proliferation, migration and tube formation of CM endothelial cells. VEGF/VEGFR signaling pathways play important regulating roles in CM angiogenesis.

  19. Corneal Fibroblasts as Sentinel Cells and Local Immune Modulators in Infectious Keratitis

    Directory of Open Access Journals (Sweden)

    Ken Fukuda

    2017-08-01

    Full Text Available The cornea serves as a barrier to protect the eye against external insults including microbial pathogens and antigens. Bacterial infection of the cornea often results in corneal melting and scarring that can lead to severe visual impairment. Not only live bacteria but also their components such as lipopolysaccharide (LPS of Gram-negative bacteria contribute to the development of inflammation and subsequent corneal damage in infectious keratitis. We describe the important role played by corneal stromal fibroblasts (activated keratocytes as sentinel cells, immune modulators, and effector cells in infectious keratitis. Corneal fibroblasts sense bacterial infection through Toll-like receptor (TLR–mediated detection of a complex of LPS with soluble cluster of differentiation 14 (CD14 and LPS binding protein present in tear fluid. The cells then initiate innate immune responses including the expression of chemokines and adhesion molecules that promote the recruitment of inflammatory cells necessary for elimination of the infecting bacteria. Infiltrated neutrophils are activated by corneal stromal collagen and release mediators that stimulate the production of pro–matrix metalloproteinases by corneal fibroblasts. Elastase produced by Pseudomonas aeruginosa (P. aeruginosa activates these released metalloproteinases, resulting in the degradation of stromal collagen. The modulation of corneal fibroblast activation and of the interaction of these cells with inflammatory cells and bacteria is thus important to minimize corneal scarring during treatment of infectious keratitis. Pharmacological agents that are able to restrain such activities of corneal fibroblasts without allowing bacterial growth represent a potential novel treatment option for prevention of excessive scarring and tissue destruction in the cornea.

  20. Polysaccharide coating of human corneal endothelium

    DEFF Research Database (Denmark)

    Schroder, H D; Sperling, S

    1977-01-01

    Electron microscopy revealed the presence of a 600-1500 A thick layer of polysaccharide on the surface of human corneal endothelial cells. The surface layer was visualized by combined fixation and staining in a mixture of ruthenium red and osmium tetroxide. The coating material was stable...

  1. Mesenchymal stem cells: Potential role in corneal wound repair and transplantation

    Institute of Scientific and Technical Information of China (English)

    Fei; Li; Shao-Zhen; Zhao

    2014-01-01

    Corneal diseases are a major cause of blindness in the world. Although great progress has been achieved in the treatment of corneal diseases, wound healing after severe corneal damage and immunosuppressive therapy after corneal transplantation remain prob-lematic. Mesenchymal stem cells(MSCs) derived from bone marrow or other adult tissues can differentiate into various types of mesenchymal lineages, such as osteocytes, adipocytes, and chondrocytes, both in vivo and in vitro. These cells can further differentiate into specific cell types under specific conditions. MSCs migrate to injury sites and promote wound healing by secreting anti-inflammatory and growth factors. In ad-dition, MSCs interact with innate and acquired immune cells and modulate the immune response through their powerful paracrine function. Over the last decade, MSCs have drawn considerable attention because of their beneficial properties and promising therapeutic prospective. Furthermore, MSCs have been applied to various studies related to wound healing, autoim-mune diseases, and organ transplantation. This review discusses the potential functions of MSCs in protecting corneal tissue and their possible mechanisms in corneal wound healing and corneal transplantation.

  2. Arecoline is cytotoxic for human endothelial cells.

    Science.gov (United States)

    Ullah, Mafaz; Cox, Stephen; Kelly, Elizabeth; Boadle, Ross; Zoellner, Hans

    2014-11-01

    Oral submucous fibrosis is a pre-malignant fibrotic condition caused by areca nut use and involves reduced mucosal vascularity. Arecoline is the principal areca nut alkaloid and is cytotoxic for epithelium and fibroblasts. Endothelial cell cycle arrest is reported on exposure to arecoline, as is cytotoxicity for endothelial-lung carcinoma hybrid cells. We here describe cytotoxicity for primary human endothelial cultures from seven separate donors. Human umbilical vein endothelial cells were exposed to increasing concentrations of arecoline and examined by: phase-contrast microscopy, haemocytometer counts, transmission electron microscopy, lactate dehydrogenase release and the methyl-thiazol-tetrazolium assay. Vacuolation and detachment of endothelium were observed at and above arecoline concentrations of 333 μg/ml or more. Ultrastructural features of cellular stress were seen after 24-h treatment with 111 μg/ml arecoline and included reduced ribosomal studding of endoplasmic reticulum, increased autophagolysosomal structures, increased vacuolation and reduced mitochondrial cristae with slight swelling. Similar changes were seen at 4 h with arecoline at 333 μg/ml or above, but with more severe mitochondrial changes including increased electron density of mitochondrial matrix and greater cristal swelling, while by 24 h, these cells were frankly necrotic. Haemocytometer counts were paralleled by both lactate dehydrogenase release and the methyl-thiazol-tetrazolium assays. Arecoline is cytotoxic via necrosis for endothelium, while biochemical assays indicate no appreciable cellular leakage before death and detachment, as well as no clear effect on mitochondrial function in viable cells. Arecoline toxicity may thus contribute to reduced vascularity in oral submucous fibrosis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. [The causes of necrobiosis and apoptosis of corneal epithelial cells during primary acquired keratoconus].

    Science.gov (United States)

    Ziangirova, G G; Antonova, O V

    2002-01-01

    We studied 56 biopsy samples of conjunctiva and 50 corneal discs excised from 28 patients with acquired keratoconus cornea. The conjunctivas in all biopsy samples showed various stages of immune inflammation. Necrobiotic changes have been revealed in epithelium of the corneal discs going by the pathways of apoptosis--programmed cell death--and oncosis--initial edematic stage of necrobiosis. At the stage of acute inflammation they are due to cytotoxic effect of the lymphocytes, monocytes, and macrophages. Antibody-dependent cytotoxicity mediated by plasma and lymphoid cells predominates at this stage. At the reparative stage of inflammation ischemia, an inductor of apoptosis and oncosis, underlies necrobiotic changes in corneal epithelium.

  4. Adhesion of endothelial cells and endothelial progenitor cells on peptide-linked polymers in shear flow.

    Science.gov (United States)

    Wang, Xin; Cooper, Stuart

    2013-05-01

    The initial adhesion of human umbilical vein endothelial cells (HUVECs), cord blood endothelial colony-forming cells (ECFCs), and human blood outgrowth endothelial cells (HBOECs) was studied under radial flow conditions. The surface of a variable shear-rate device was either coated with polymer films or covered by synthetic fibers. Spin-coating was applied to produce smooth polymer films, while fibrous scaffolds were generated by electrospinning. The polymer was composed of hexyl methacrylate, methyl methacrylate, poly(ethylene glycol) methacrylate (PEGMA), and CGRGDS peptide. The peptide was incorporated into the polymer system by coupling to an acrylate-PEG-N-hydroxysuccinimide comonomer. A shear-rate-dependent increase of the attached cells with time was observed with all cell types. The adhesion of ECs increased on RGD-linked polymer surfaces compared to polymers without adhesive peptides. The number of attached ECFCs and HBOECs are significantly higher than that of HUVECs within the entire shear-rate range and surfaces examined, especially on RGD-linked polymers at low shear rates. Their superior adhesion ability of endothelial progenitor cells under flow conditions suggests they are a promising source for in vivo seeding of vascular grafts and shows the potential to be used for self-endothelialized implants.

  5. Amyloid β induces adhesion of erythrocytes to endothelial cells and affects endothelial viability and functionality.

    Science.gov (United States)

    Nakagawa, Kiyotaka; Kiko, Takehiro; Kuriwada, Satoko; Miyazawa, Taiki; Kimura, Fumiko; Miyazawa, Teruo

    2011-01-01

    It has been suggested that amyloid β-peptide (Aβ) might mediate the adhesion of erythrocytes to the endothelium which could disrupt the properties of endothelial cells. We provide evidence here that Aβ actually induced the binding of erythrocytes to endothelial cells and decreased endothelial viability, perhaps by the generation of oxidative and inflammatory stress. These changes are likely to contribute to the pathogenesis of Alzheimer's disease.

  6. The Active Metabolite of Leflunomide A771726 Inhibits Corneal Neovascularization

    Institute of Scientific and Technical Information of China (English)

    Mingchang ZHANG; Nian HAO; Fang BIAN

    2008-01-01

    The effects of A771726, the active metabolite of leflunomide, on experimental rat corneal neovascularization (NV) in vivo and on cultured human umbilical vein endothelial cells in vitro were studied. The corneal NV was induced by alkali burn in 40 SD rats. The rats were randomly divided into 4 groups with 10 rats in each group. Group A was treated with 0.9% sodium chloride (control group), and group B, group C and group D were given different concentrations of A771726 eye drops (0.5%,l.0%,2.0% respectively) 4 times daily during days 0-28. The occurrence and development of corneal NV were observed at 4,7,14,21 and 28 day after alkali burn by a slit lamp microscope. The cultured human umbilical vein endothelial cells (ECV-304) were incubated with A771726 solution at different concentrations (20,40,80,160,320μmol/L) for 36h. The proliferation of cells was assessed by methyl thiazolyl tetrazolium (MTT), and the expression of proliferating cell nuclear antigen (PCNA) in cells was detected by using immunofluorescence under the laser confocal microscope. The rat model showed that the onset of corneal NV was delayed and progression of corneal NV was inhibited in the groups C and D. The corneal NV areas in groups C and D were significantly smaller than in groups A and B (P0.05). A771726 solution (≥40μmol/L) could inhibit proliferation of human umbilical vein endothelial cells and decrease the expression of PCNA in cells significantly. A771726, as the active metabolite of leflunomide, strongly prevented corneal NV induced by alkali burn in the in vivo model, and inhibited proliferation of human umbilical vein endothelial cells in the in vitro model. Therefore, A771726 may serve as an angiogenic inhibitor in the treatment of corneal NV.

  7. Functional genomics of vascular endothelial cells

    OpenAIRE

    Wallgard, Elisabet

    2008-01-01

    Angiogenesis, the formation of new blood vessels from preexisting ones, is a process involved in normal development as well as in several pathological conditions, such as cancer, ischemic heart disease, wound healing and certain retinal complications. Antiangiogenic targeting is therefore a promising new therapeutic principle. However, few blood vessel-specific drug targets have been identified, and information is still limited about endothelial cell (EC)-specific molecular ...

  8. Long‐term recovery of the human corneal endothelium after toxic injury by benzalkonium chloride

    Science.gov (United States)

    Hughes, E H; Pretorius, M; Eleftheriadis, H; Liu, C S C

    2007-01-01

    Introduction The inadvertent intra‐ocular administration of benzalkonium chloride‐preserved hydroxypropyl methylcellulose during cataract surgery at another hospital in 1999 resulted in toxic corneal endothelial injury and profound postoperative corneal oedema as a result of endothelial decompensation. The long‐term effect of this adverse event was assessed. Methods All 19 patients were invited to return for examination including corneal endothelial specular microscopy and pachymetry seven years after the incident. Results were compared with data from one year after the incident. Results Five patients attended for examination, one had received a penetrating keratoplasty and was, therefore, excluded. Ten patients had died and four had moved out of the region and were unable to attend. All four study patients were pain free and achieved 6/12 or better. Mean central corneal thickness reduced by 13% from 652.6 μm at one year to 563.4 μm. Mean central corneal endothelial cell density (n  =  3) increased 28% from 663.7 cells/mm2 at one year to 835.7 cells/mm2 (p<0.05). Conclusions After toxic injury, corneal endothelial function may have a remarkable capacity for recovery even after the first postoperative year. The rise in central endothelial cell density may represent cell migration from less affected areas or cellular proliferation. Should this unfortunate event recur, clinicians may expect continued recovery beyond one year. PMID:17504856

  9. Application value of corneal limbus stem cells transplantation to treat pterygium

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    Miao-Ying Zhang

    2017-02-01

    Full Text Available AIM: To analyze the application value of corneal limbus stem cells transplantation in the treatment of pterygium. METHODS: Totally 300 patients(318 eyeswith pterygium were divided into 2 groups according to treatment methods. Patients of the control group(70 patients with 77 eyeswere given simple surgical resection, while patients of the observation group(230 patients with 241 eyeswere given corneal limbus stem cells transplantation after surgical resection. The postoperative recurrent rate, corneal epithelium incision healing time, corneal epithelium healing degree, pain score and tear film break-up time were observed. RESULTS: The corneal epithelium incision healing time of the observation group and control group were respectively 5.2±1.9d and 6.4±1.7d, and the difference had statistical significance(PPP>0.05. At 2wk after treatment, the corneal epithelium healing degree of the observation group was better, and the pain score of the observation group was lower than those of the control group, the differences between groups had statistical significance(PP>0.05. At 1 and 2wk after treatment, the tear film break-up time of the observation group was longer than that of the control group(PCONCLUSION: Corneal limbus stem cell transplantation can prevent postoperative recurrence of pterygium effectively, and its postoperative tissue repair effect is superior to simple surgery.

  10. Identification of human fibroblast cell lines as a feeder layer for human corneal epithelial regeneration.

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    Rong Lu

    Full Text Available There is a great interest in using epithelium generated in vitro for tissue bioengineering. Mouse 3T3 fibroblasts have been used as a feeder layer to cultivate human epithelia including corneal epithelial cells for more than 3 decades. To avoid the use of xeno-components, we evaluated human fibroblasts as an alternative feeder supporting human corneal epithelial regeneration. Five human fibroblast cell lines were used for evaluation with mouse 3T3 fibroblasts as a control. Human epithelial cells isolated from fresh corneal limbal tissue were seeded on these feeders. Colony forming efficiency (CFE and cell growth capacity were evaluated on days 5-14. The phenotype of the regenerated epithelia was evaluated by morphology and immunostaining with epithelial markers. cDNA microarray was used to analyze the gene expression profile of the supportive human fibroblasts. Among 5 strains of human fibroblasts evaluated, two newborn foreskin fibroblast cell lines, Hs68 and CCD1112Sk, were identified to strongly support human corneal epithelial growth. Tested for 10 passages, these fibroblasts continually showed a comparative efficiency to the 3T3 feeder layer for CFE and growth capacity of human corneal epithelial cells. Limbal epithelial cells seeded at 1 × 10(4 in a 35-mm dish (9.6 cm(2 grew to confluence (about 1.87-2.41 × 10(6 cells in 12-14 days, representing 187-241 fold expansion with over 7-8 doublings on these human feeders. The regenerated epithelia expressed K3, K12, connexin 43, p63, EGFR and integrin β1, resembling the phenotype of human corneal epithelium. DNA microarray revealed 3 up-regulated and 10 down-regulated genes, which may be involved in the functions of human fibroblast feeders. These findings demonstrate that commercial human fibroblast cell lines support human corneal epithelial regeneration, and have potential use in tissue bioengineering for corneal reconstruction.

  11. Tissue engineering of corneal stromal layer with dermal fibroblasts: phenotypic and functional switch of differentiated cells in cornea.

    Science.gov (United States)

    Zhang, Yan Qing; Zhang, Wen Jie; Liu, Wei; Hu, Xiao Jie; Zhou, Guang Dong; Cui, Lei; Cao, Yilin

    2008-02-01

    Previously, we successfully engineered a corneal stromal layer using corneal stromal cells. However, the limited source and proliferation potential of corneal stromal cells has driven us to search for alternative cell sources for corneal stroma engineering. Based on the idea that the tissue-specific environment may alter cell fate, we proposed that dermal fibroblasts could switch their phenotype to that of corneal stromal cells in the corneal environment. Thus, dermal fibroblasts were harvested from newborn rabbits, seeded on biodegradable polyglycolic acid (PGA) scaffolds, cultured in vitro for 1 week, and then implanted into adult rabbit corneas. After 8 weeks of implantation, nearly transparent corneal stroma was formed, with a histological structure similar to that of its native counterpart. The existence of cells that had been retrovirally labeled with green fluorescence protein (GFP) demonstrated the survival of implanted cells. In addition, all GFP-positive cells that survived expressed keratocan, a specific marker for corneal stromal cells, and formed fine collagen fibrils with a highly organized pattern similar to that of native stroma. However, neither dermal fibroblast-PGA construct pre-incubated in vitro for 3 weeks nor chondrocyte-PGA construct could form transparent stroma. The results demonstrated that neonatal dermal fibroblasts could switch their phenotype in the new tissue environment under restricted conditions. The functional restoration of corneal transparency using dermal fibroblasts suggests that they could be an alternative cell source for corneal stroma engineering.

  12. Técnica de separação da membrana de Descemet para transplante de células endoteliais da córnea: estudo experimental em coelhos Technique for separating Descemet membrane for corneal endothelial cells transplantation: experimental study in rabbits

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    Daniel Wasilewski

    2010-02-01

    Full Text Available OBJETIVO: Avaliar a porcentagem de dano endotelial induzido por uma técnica cirúrgica para a separação da membrana de Descemet contendo endotélio sadio, analisar a viabilidade e eficácia desta técnica, e avaliar a porcentagem de dano endotelial causado pela inversão da córnea em câmara anterior artificial. MÉTODOS: As córneas de três grupos de 12 coelhos da linhagem Nova Zelândia foram avaliadas. O grupo 1 foi usado como controle; portanto, as córneas foram analisadas após coletadas e trepanadas. O grupo 2 foi analisado após a inversão da córnea (endotélio para cima na posição convexa, montada em câmara anterior artificial, para o cálculo da porcentagem do dano endotelial induzido por esta inversão. O grupo 3 foi avaliado após a separação entre a membrana de Descemet e o estroma com o uso de substância viscoelástica em córneas invertidas e montadas em câmara anterior artificial. O dano endotelial foi avaliado por meio de fotografias digitais tiradas no microscópio após impregnar o endotélio com vermelho de alizarina. Amostras do grupo 3 foram processadas para avaliação histopatológica. RESULTADOS: O grupo 3 (separação viscoelástica apresentou um índice de lesão celular endotelial de 10,06%, o grupo 2 apresentou um índice de 3,58% e o grupo controle um índice de 0,18% de lesão celular endotelial (pPURPOSE: To evaluate the percentage of endothelial cell damage induced during a surgical technique of Descemet's membrane separation containing healthy endothelium, analyze the viability and efficacy of this technique, and evaluate the percentage of endothelial cell damage caused by inversion of the cornea on an artificial anterior chamber. METHODS: The corneas from three groups of 12 New Zealand rabbits were evaluated. The Group one was used as the control, so the corneas were analyzed after collected and trephinated. The Group two was analyzed after inversion of the cornea (endothelial side up at a convex

  13. The cytokine regulation of SPARC production by rabbit corneal epithelial cells and fibroblasts in vitro.

    Science.gov (United States)

    Abe, Kosuke; Hibino, Tsuyoshi; Mishima, Hiroshi; Shimomura, Yoshikazu

    2004-03-01

    SPARC (osteonectin/BM40) is detected in the corneal stroma during the wound-healing process. To understand the metabolism of SPARC in the cornea, we investigated the effects of cytokines and growth factors on SPARC synthesis by rabbit corneal epithelial cells and fibroblasts. Rabbit corneal epithelial cells or fibroblasts were cultured for 3 days with serum-containing minimal essential medium (MEM), then subcultured for 3 days on serum-free MEM with epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta), or interleukin-1beta (IL-1beta). SPARC concentration in the medium was measured by the ELISA method using anti-SPARC monoclonal antibody. The concentration of SPARC in the conditioned medium of the epithelial cells depended on either cell numbers or cultivation periods. When EGF was added to the medium, the amount of SPARC in the medium decreased. The addition of IL-1beta, PDGF, or TGF-beta did not affect SPARC synthesis by the epithelial cells. The production of SPARC by rabbit corneal fibroblasts was low compared with that by epithelial cells. However, the synthesis of SPARC by corneal fibroblasts was significantly enhanced by the addition of TGF-beta. The addition of IL-1beta, PDGF, or EGF slightly increased SPARC synthesis by corneal fibroblasts. Cytokines and growth factors modulate SPARC synthesis by rabbit corneal epithelial cells and fibroblasts. These results suggest that cytokines and growth factors modulate cell-matrix interaction in corneal wound healing, possibly by regulating SPARC synthesis.

  14. Generation and evaluation of a human corneal model cell system for ophthalmologic issues using the HPV16 E6/E7 oncogenes as uniform immortalization platform.

    Science.gov (United States)

    Schulz, Simon; Steinberg, Thorsten; Beck, David; Tomakidi, Pascal; Accardi, Rosita; Tommasino, Massimo; Reinhard, Thomas; Eberwein, Philipp

    2013-01-01

    The present study aimed at employing the human papillomavirus type 16 (HPV16) E6/E7 gene platform, to create a uniform authentic in vitro model cell system of the human cornea for ophthalmologic issues and here especially for prospective biomaterial evaluations for therapeutic regenerative approaches. Therefore, HPV16 E6/E7 genes were employed as uniform platform to immortalize primary human corneal keratinocytes (IHCK), fibroblasts (IHCF), and endothelial (IHCE) cells. qPCR revealed that E6/E7 mRNA transcription persisted at rising passages and FISH detection of the chromosome portfolio 1, 8, 10 and 18 showed fairly the disomic cytogenetic status. Hot spot passages proved oscillation of aneuploidies in the entire passage spectrum under study, while hot spot aneuploidies annotated prevalence for distinct chromosomes. Though IIF revealed general endurance, tissue-innate corneal biomarkers were modulated, i.e. expressed in a temporal-confluence, temporal-spatial or passage-dependent manner. In summary, by the fairly normal chromosomal status, and expression of tissue-innate biomarkers, we created for the first time a uniform authentic in vitro model cell system of the human cornea, by application of the HPV16 E6/E7 immortalization platform only. This system renders a precious tool for prospective iterative in vitro studies on issues such as corneal tissue homeostasis, pharmaceutical generics, and/or evaluation of new biomaterials for clinical corneal applications. Copyright © 2013 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  15. Adipose-derived mesenchymal stem cell administration does not improve corneal graft survival outcome.

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    Sherezade Fuentes-Julián

    Full Text Available The effect of local and systemic injections of mesenchymal stem cells derived from adipose tissue (AD-MSC into rabbit models of corneal allograft rejection with either normal-risk or high-risk vascularized corneal beds was investigated. The models we present in this study are more similar to human corneal transplants than previously reported murine models. Our aim was to prevent transplant rejection and increase the length of graft survival. In the normal-risk transplant model, in contrast to our expectations, the injection of AD-MSC into the graft junction during surgery resulted in the induction of increased signs of inflammation such as corneal edema with increased thickness, and a higher level of infiltration of leukocytes. This process led to a lower survival of the graft compared with the sham-treated corneal transplants. In the high-risk transplant model, in which immune ocular privilege was undermined by the induction of neovascularization prior to graft surgery, we found the use of systemic rabbit AD-MSCs prior to surgery, during surgery, and at various time points after surgery resulted in a shorter survival of the graft compared with the non-treated corneal grafts. Based on our results, local or systemic treatment with AD-MSCs to prevent corneal rejection in rabbit corneal models at normal or high risk of rejection does not increase survival but rather can increase inflammation and neovascularization and break the innate ocular immune privilege. This result can be partially explained by the immunomarkers, lack of immunosuppressive ability and immunophenotypical secretion molecules characterization of AD-MSC used in this study. Parameters including the risk of rejection, the inflammatory/vascularization environment, the cell source, the time of injection, the immunosuppression, the number of cells, and the mode of delivery must be established before translating the possible benefits of the use of MSCs in corneal transplants to clinical

  16. IL-8 and MCP Gene Expression and Production by LPS-Stimulated Human Corneal Stromal Cells

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    Roni M. Shtein

    2012-01-01

    Full Text Available Purpose. To determine time course of effect of lipopolysaccharide (LPS on production of interleukin-8 (IL-8 and monocyte chemotactic protein (MCP by cultured human corneal stromal cells. Methods. Human corneal stromal cells were harvested from donor corneal specimens, and fourth to sixth passaged cells were used. Cell cultures were stimulated with LPS for 2, 4, 8, and 24 hours. Northern blot analysis of IL-8 and MCP gene expression and ELISA for IL-8 and MCP secretion were performed. ELISA results were analyzed for statistical significance using two-tailed Student's t-test. Results. Northern blot analysis demonstrated significantly increased IL-8 and MCP gene expression after 4 and 8 hours of exposure to LPS. ELISA for secreted IL-8 and MCP demonstrated statistically significant increases (P<0.05 after corneal stromal cell stimulation with LPS. Conclusions. This paper suggests that human corneal stromal cells may participate in corneal inflammation by secreting potent leukocyte chemotactic and activating proteins in a time-dependent manner when exposed to LPS.

  17. Investigation the Porous Collagen-Chitosan /Glycosaminoglycans for Corneal Cell Culture as Tissue Engineering Scaffold

    Institute of Scientific and Technical Information of China (English)

    LI Qin-Hua; CHEN Jian-Su

    2005-01-01

    The objective of this study was to produce the porous collagen-chitosan/Glycosanminglycans (GAG) for corneal ceil-seed implant as a three-dimensional tissue engineering scaffold to improve the regeneration corneas. The effect of various content of glycerol as form porous agent to collagen-chitosan/GAG preserved a porous dimensional structure was investigated. The heat-drying was used to prepare porous collagen-chitosan /GAG scaffold. The pore morphology of collagenchitosan/GAG was controlled by changing the concentration of glycerol solution and drying methods. The porous structure morphology was observed by SEM. The diameter of the pores form 10 to 50 μm. The highly porous scaffold had interconnecting pores. The corneal cell morphology was observed under the light microscope. These results suggest that collagen-chitosan/GAG showed that corneal cell have formed confluent layers and resemble the surface of normal corneal cell surface.

  18. Endothelial progenitor cells with Alzheimer's disease

    Institute of Scientific and Technical Information of China (English)

    KONG Xiao-dong; ZHANG Yun; LIU Li; SUN Ning; ZHANG Ming-yi; ZHANG Jian-ning

    2011-01-01

    Background Endothelial dysfunction is thought to be critical events in the pathogenesis of Alzheimer's disease (AD).Endothelial progenitor cells (EPCs) have provided insight into maintaining and repairing endothelial function. To study the relation between EPCs and AD, we explored the number of circulating EPCs in patients with AD.Methods A total of 104 patients were recruited from both the outpatients and inpatients of the geriatric neurology department at General Hospital, rianjin Medical University. Consecutive patients with newly diagnosed AD (n=30),patients with vascular dementia (VaD, n=34), and healthy elderly control subjects with normal cognition (n=40) were enrolled after matching for age, gender, body mass index, medical history, current medication and Mini Mental State Examination. Middle cerebral artery flow velocity was examined with transcranial Doppler. Endothelial function was evaluated according to the level of EPCs, and peripheral blood EPCs was counted by flow cytometry.Results There were no significant statistical differences of clinical data in AD, VaD and control groups (P >0.05). The patients with AD showed decreased CD34-positive (CD34+) or CD133-positive (CD133+) levels compared to the control subjects, but there were no significant statistical differences in patients with AD. The patients with AD had significantly lower CD34+CD133+ EPCs(CD34 and CD133 double positive endothelial progenitor cells) than the control subjects (P <0.05). In the patients with AD, a lower CD34+CD133+ EPCs count was independently associated with a lower Mini-Mental State Examination score (r=0.514, P=0.004). Patients with VaD also showed a significant decrease in CD34+CD133+ EPCs levels, but this was not evidently associated with the Mini-Mental State Examination score. The changes of middle cerebral artery flow velocity were similar between AD and VaD. Middle cerebral artery flow velocity was decreased in the AD and VaD groups and significantly lower than

  19. NK cells are necessary for recovery of corneal CD11c+ dendritic cells after epithelial abrasion injury

    Science.gov (United States)

    Mechanisms controlling CD11c(+) MHCII(+) DCs during corneal epithelial wound healing were investigated in a murine model of corneal abrasion. Selective depletion of NKp46(+) CD3- NK cells that normally migrate into the cornea after epithelial abrasion resulted in >85% reduction of the epithelial CD1...

  20. The graft of autologous adipose-derived stem cells in the corneal stromal after mechanic damage.

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    Xiao-Yun Ma

    Full Text Available This study was designed to explore the feasibility of using autologous rabbit adipose derived stem cells (rASCs as seed cells and polylactic-co-glycolic acid (PLGA as a scaffold for repairing corneal stromal defects. rASCs isolated from rabbit nape adipose tissue were expanded and seeded on a PLGA scaffold to fabricate cell-scaffold constructs. After 1 week of cultivation in vitro, the cell-scaffold complexes were transplanted into corneal stromal defects in rabbits. In vivo, the autologous rASCs-PLGA constructed corneal stroma gradually became transparent without corneal neovascularization after 12 weeks. Hematoxylin and eosin staining and transmission electron microscopy examination revealed that their histological structure and collagen fibril distribution at 24 weeks after implantation were similar to native counterparts. As to the defect treated with PLGA alone, the stromal defects remained. And scar tissue was observed in the untreated-group. The implanted autologous ASCs survived up to 24 weeks post-transplantation and differentiated into functional keratocytes, as assessed by the expression of aldehyde-3-dehydrogenase1A1 (ALDH1A1 and cornea-specific proteoglycan keratocan. Our results revealed that autologous rASCs could be one of the cell sources for corneal stromal restoration in diseased corneas or for tissue engineering of a corneal equivalent.

  1. Changes in corneal endothelium cell characteristics after cataract surgery with and without use of viscoelastic substances during intraocular lens implantation

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    Schulze SD

    2015-11-01

    Full Text Available Stephan D Schulze,1 Thomas Bertelmann,1 Irena Manojlovic,2 Stefan Bodanowitz,2 Sebastian Irle,3 Walter Sekundo11Department of Ophthalmology, Philipps University of Marburg, Marburg, 2Private Practice and Ambulatory Surgical Center, Bremen, 3Freelance Statistician, Friedberg, GermanyPurpose: To evaluate whether the use of balanced salt solution (BSS or an ophthalmic viscoelastic device (OVD during hydrophilic acrylic intraocular lens (IOL implantation variously impacts corneal endothelial cell characteristics in eyes undergoing uneventful phacoemulsifications.Methods: Prospective nonrandomized observational clinical trial. Patients were assigned either to the BSS plus® or to the OVD Z-Celcoat™ group depending on the substance used during IOL implantation. Corneal endothelium cell characteristics were obtained before, 1 week, and 6 weeks after surgery. Intraoperative parameters (eg, surgery time, phacoemulsification energy were recorded.Results: Ninety-seven eyes were assigned to the BSS plus and 86 eyes to the Z-Celcoat group. Preoperative corneal endothelium cell density (ECD and endothelium cell size were 2,506±310 cells/mm2/2,433±261 cells/mm2 and 406±47 µm2/416±50 µm2 (P=0.107/P=0.09. After 1 and 6 weeks, ECD decreased and endothelium cell size increased significantly in both groups (each P<0.001 without significant differences between both groups (each P>0.05. Irrigation–aspiration suction time (30.3±16.6 versus 36.3±14.5 seconds and overall surgical time (7.2±1.2 versus 8.0±1.4 minutes were significantly longer in the OVD Z-Celcoat group (each P<0.001. No complications or serious side effects occurred.Conclusion: Implantation of a hydrophilic acrylic IOL under BSS infusion seems to be a useful and faster alternative in experienced hands without generating higher ECD loss rates.Keywords: phacoemulsification, ophthalmic viscoelastic device, endothelial cell density, IOL

  2. Corneal cell adhesion to contact lens hydrogel materials enhanced via tear film protein deposition.

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    Claire M Elkins

    Full Text Available Tear film protein deposition on contact lens hydrogels has been well characterized from the perspective of bacterial adhesion and viability. However, the effect of protein deposition on lens interactions with the corneal epithelium remains largely unexplored. The current study employs a live cell rheometer to quantify human corneal epithelial cell adhesion to soft contact lenses fouled with the tear film protein lysozyme. PureVision balafilcon A and AirOptix lotrafilcon B lenses were soaked for five days in either phosphate buffered saline (PBS, borate buffered saline (BBS, or Sensitive Eyes Plus Saline Solution (Sensitive Eyes, either pure or in the presence of lysozyme. Treated contact lenses were then contacted to a live monolayer of corneal epithelial cells for two hours, after which the contact lens was sheared laterally. The apparent cell monolayer relaxation modulus was then used to quantify the extent of cell adhesion to the contact lens surface. For both lens types, lysozyme increased corneal cell adhesion to the contact lens, with the apparent cell monolayer relaxation modulus increasing up to an order of magnitude in the presence of protein. The magnitude of this increase depended on the identity of the soaking solution: lenses soaked in borate-buffered solutions (BBS, Sensitive Eyes exhibited a much greater increase in cell attachment upon protein addition than those soaked in PBS. Significantly, all measurements were conducted while subjecting the cells to moderate surface pressures and shear rates, similar to those experienced by corneal cells in vivo.

  3. Endothelial cells, tissue factor and infectious diseases

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    Lopes-Bezerra L.M.

    2003-01-01

    Full Text Available Tissue factor is a transmembrane procoagulant glycoprotein and a member of the cytokine receptor superfamily. It activates the extrinsic coagulation pathway, and induces the formation of a fibrin clot. Tissue factor is important for both normal homeostasis and the development of many thrombotic diseases. A wide variety of cells are able to synthesize and express tissue factor, including monocytes, granulocytes, platelets and endothelial cells. Tissue factor expression can be induced by cell surface components of pathogenic microorganisms, proinflammatory cytokines and membrane microparticles released from activated host cells. Tissue factor plays an important role in initiating thrombosis associated with inflammation during infection, sepsis, and organ transplant rejection. Recent findings suggest that tissue factor can also function as a receptor and thus may be important in cell signaling. The present minireview will focus on the role of tissue factor in the pathogenesis of septic shock, infectious endocarditis and invasive aspergillosis, as determined by both in vivo and in vitro models.

  4. Production of soluble Neprilysin by endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kuruppu, Sanjaya, E-mail: Sanjaya.Kuruppu@monash.edu [Department of Biochemistry and Molecular Biology, Building 77, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia); Rajapakse, Niwanthi W. [Department of Physiology, Building 13F, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia); Minond, Dmitriy [Torrey Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port Saint Lucie, FL 34987 (United States); Smith, A. Ian [Department of Biochemistry and Molecular Biology, Building 77, Monash University, Wellington Rd, Clayton, Vic 3800 (Australia)

    2014-04-04

    Highlights: • A soluble full-length form of Neprilysin exists in media of endothelial cells. • Exosomal release is the key mechanism for the production of soluble Neprilysin. • Inhibition of ADAM-17 by specific inhibitors reduce Neprilysin release. • Exosome mediated release of Neprilysin is dependent on ADAM-17 activity. - Abstract: A non-membrane bound form of Neprilysin (NEP) with catalytic activity has the potential to cleave substrates throughout the circulation, thus leading to systemic effects of NEP. We used the endothelial cell line Ea.hy926 to identify the possible role of exosomes and A Disintegrin and Metalloprotease 17 (ADAM-17) in the production of non-membrane bound NEP. Using a bradykinin based quenched fluorescent substrate (40 μM) assay, we determined the activity of recombinant human NEP (rhNEP; 12 ng), and NEP in the media of endothelial cells (10% v/v; after 24 h incubation with cells) to be 9.35 ± 0.70 and 6.54 ± 0.41 μmols of substrate cleaved over 3 h, respectively. The presence of NEP in the media was also confirmed by Western blotting. At present there are no commercially available inhibitors specific for ADAM-17. We therefore synthesised two inhibitors TPI2155-14 and TPI2155-17, specific for ADAM-17 with IC{sub 50} values of 5.36 and 4.32 μM, respectively. Treatment of cells with TPI2155-14 (15 μM) and TPI2155-17 (4.3 μM) resulted in a significant decrease in NEP activity in media (62.37 ± 1.43 and 38.30 ± 4.70, respectively as a % of control; P < 0.0001), implicating a possible role for ADAM-17 in NEP release. However, centrifuging media (100,000g for 1 h at 4 °C) removed all NEP activity from the supernatant indicating the likely role of exosomes in the release of NEP. Our data therefore indicated for the first time that NEP is released from endothelial cells via exosomes, and that this process is dependent on ADAM-17.

  5. Endothelial Progenitor Cells Enter the Aging Arena.

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    Kate eWilliamson

    2012-02-01

    Full Text Available Age is a significant risk factor for the development of vascular diseases, such as atherosclerosis. Although pharmacological treatments, including statins and anti-hypertensive drugs, have improved the prognosis for patients with cardiovascular disease, it remains a leading cause of mortality in those aged 65 years and over. Furthermore, given the increased life expectancy of the population in developed countries, there is a clear need for alternative treatment strategies. Consequently, the relationship between aging and progenitor cell-mediated repair is of great interest. Endothelial progenitor cells (EPCs play an integral role in the cellular repair mechanisms for endothelial regeneration and maintenance. However, EPCs are subject to age-associated changes that diminish their number in circulation and function, thereby enhancing vascular disease risk. A great deal of research is aimed at developing strategies to harness the regenerative capacity of these cells.In this review, we discuss the current understanding of the cells termed ‘EPCs’, examine the impact of age on EPC-mediated repair and identify therapeutic targets with potential for attenuating the age-related decline in vascular health via beneficial actions on EPCs.

  6. Enhancing endothelial progenitor cell for clinical use

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Circulating endothelial progenitor cells (EPCs) havebeen demonstrated to correlate negatively with vascularendothelial dysfunction and cardiovascular risk factors.However, translation of basic research into the clinicalpractice has been limited by the lack of unambiguousand consistent definitions of EPCs and reduced EPCcell number and function in subjects requiring them forclinical use. This article critically reviews the definitionof EPCs based on commonly used protocols, their valueas a biomarker of cardiovascular risk factor in subjectswith cardiovascular disease, and strategies to enhanceEPCs for treatment of ischemic diseases.

  7. Corneal Cross-Linking (with a Partial Deepithelization) in Keratoconus with Five Years of Follow-Up

    Science.gov (United States)

    Galvis, Virgilio; Tello, Alejandro; Carreño, Néstor I.; Ortiz, Alvaro I.; Barrera, Rodrigo; Rodriguez, Carlos Julián; Ochoa, Miguel E.

    2016-01-01

    We performed a retrospective interventional case series including 80 eyes of 48 patients with keratoconus (KC) who were treated with modified corneal cross-linking (CXL) for KC (with a partial deepithelization in a pattern of stripes). The average follow-up was 5.8 years (with a minimum of 5 years). At the last follow-up visit, compared with preoperative values, there were no significant changes in spherical equivalent, average keratometry, corneal thickness, corneal hysteresis, or corneal resistance factor. The distance-corrected visual acuity was 20/39 preoperatively and 20/36 postoperatively (P = 0.3). The endothelial cell count decreased by 4.7% (P < 0.005). These findings suggest that this modified corneal CXL technique is a safe and effective alternative to halt the progression of KC up to five years after the procedure. However, some concerns remain as to whether this technique can affect in some degree the corneal endothelial cells. PMID:27199574

  8. Primary corneal papilloma and squamous cell carcinoma associated with pigmentary keratitis in four dogs.

    Science.gov (United States)

    Bernays, M E; Flemming, D; Peiffer, R L

    1999-01-15

    Squamous cell carcinoma (SCC) and squamous papilloma are rarely reported as primary lesions of the cornea in dogs. One case of corneal papilloma and 3 cases of SCC, each arising as a primary central corneal neoplasm rather than spreading from adjacent limbal conjunctiva, were reviewed. The most common cause of SCC in animals is chronic exposure of lightly pigmented epithelium to UV light; however, all dogs in this study had a history of chronic pigmentary keratitis. Three of the 4 dogs were of brachycephalic breeds with naturally proptotic eyes and oversized palpebral fissures that may have exposed the cornea to greater excessive solar radiation. Alternatively, mechanical factors that caused chronic changes in the cornea may have been causative factors for induction of primary dysplastic or neoplastic changes. Primary corneal neoplasia should be considered in the differential diagnosis of corneal masses.

  9. Differentiation state determines neural effects on microvascular endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Muffley, Lara A., E-mail: muffley@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Pan, Shin-Chen, E-mail: pansc@mail.ncku.edu.tw [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Smith, Andria N., E-mail: gnaunderwater@gmail.com [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Ga, Maricar, E-mail: marga16@uw.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Hocking, Anne M., E-mail: ahocking@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Gibran, Nicole S., E-mail: nicoleg@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States)

    2012-10-01

    Growing evidence indicates that nerves and capillaries interact paracrinely in uninjured skin and cutaneous wounds. Although mature neurons are the predominant neural cell in the skin, neural progenitor cells have also been detected in uninjured adult skin. The aim of this study was to characterize differential paracrine effects of neural progenitor cells and mature sensory neurons on dermal microvascular endothelial cells. Our results suggest that neural progenitor cells and mature sensory neurons have unique secretory profiles and distinct effects on dermal microvascular endothelial cell proliferation, migration, and nitric oxide production. Neural progenitor cells and dorsal root ganglion neurons secrete different proteins related to angiogenesis. Specific to neural progenitor cells were dipeptidyl peptidase-4, IGFBP-2, pentraxin-3, serpin f1, TIMP-1, TIMP-4 and VEGF. In contrast, endostatin, FGF-1, MCP-1 and thrombospondin-2 were specific to dorsal root ganglion neurons. Microvascular endothelial cell proliferation was inhibited by dorsal root ganglion neurons but unaffected by neural progenitor cells. In contrast, microvascular endothelial cell migration in a scratch wound assay was inhibited by neural progenitor cells and unaffected by dorsal root ganglion neurons. In addition, nitric oxide production by microvascular endothelial cells was increased by dorsal root ganglion neurons but unaffected by neural progenitor cells. -- Highlights: Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell proliferation. Black-Right-Pointing-Pointer Neural progenitor cells, not dorsal root ganglion neurons, regulate microvascular endothelial cell migration. Black-Right-Pointing-Pointer Neural progenitor cells and dorsal root ganglion neurons do not effect microvascular endothelial tube formation. Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate

  10. Stiffness of polyelectrolyte multilayer film influences endothelial function of endothelial cell monolayer.

    Science.gov (United States)

    Chang, Hao; Zhang, He; Hu, Mi; Chen, Jia-Yan; Li, Bo-Chao; Ren, Ke-Feng; Martins, M Cristina L; Barbosa, Mário A; Ji, Jian

    2017-01-01

    Endothelialization has proved to be critical for maintaining long-term success of implantable vascular devices. The formation of monolayer of endothelial cells (ECs) on the implant surfaces is one of the most important factors for the endothelialization. However, endothelial function of regenerated EC monolayer, which plays a much more important role in preventing the complications of post-implantation, has not received enough attention. Here, a vascular endothelial growth factor (VEGF)-incorporated poly(l-lysine)/hyaluronan (PLL/HA) polyelectrolyte multilayer film was fabricated. Through varying the crosslinking degree, stiffness of the film was manipulated, offering either soft or stiff film. We demonstrated that ECs were able to adhere and proliferate on both soft and stiff films, subsequently forming an integrated EC monolayer. Furthermore, endothelial functions were evaluated by characterizing EC monolayer integrity, expression of genes correlated with the endothelial functions, and nitric oxide production. It demonstrated that EC monolayer on the soft film displayed higher endothelial function compared to that on the stiff film. Our study highlights the influence of substrate stiffness on endothelial function, which offers a new criterion for surface design of vascular implants. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Asiaticoside Inhibits TNF-α-Induced Endothelial Hyperpermeability of Human Aortic Endothelial Cells.

    Science.gov (United States)

    Fong, Lai Yen; Ng, Chin Theng; Zakaria, Zainul Amiruddin; Baharuldin, Mohamad Taufik Hidayat; Arifah, Abdul Kadir; Hakim, Muhammad Nazrul; Zuraini, Ahmad

    2015-10-01

    The increase in endothelial permeability often promotes edema formation in various pathological conditions. Tumor necrosis factor-alpha (TNF-α), a pro-atherogenic cytokine, impairs endothelial barrier function and causes endothelial dysfunction in early stage of atherosclerosis. Asiaticoside, one of the triterpenoids derived from Centella asiatica, is known to possess antiinflammatory activity. In order to examine the role of asiaticoside in preserving the endothelial barrier, we assessed its effects on endothelial hyperpermeability and disruption of actin filaments evoked by TNF-α in human aortic endothelial cells (HAEC). TNF-α caused an increase in endothelial permeability to fluorescein isothiocyanate (FITC)-dextran. Asiaticoside pretreatment significantly suppressed TNF-α-induced increased permeability. Asiaticoside also prevented TNF-α-induced actin redistribution by suppressing stress fiber formation. However, the increased F to G actin ratio stimulated by TNF-α was not changed by asiaticoside. Cytochalasin D, an actin depolymerizing agent, was used to correlate the anti-hyperpermeability effect of asiaticoside with actin cytoskeleton. Surprisingly, asiaticoside failed to prevent cytochalasin D-induced increased permeability. These results suggest that asiaticoside protects against the disruption of endothelial barrier and actin rearrangement triggered by TNF-α without a significant change in total actin pool. However, asiaticoside seems to work by other mechanisms to maintain the integrity of endothelial barrier rather than stabilizing the F-actin organization.

  12. Quantitative & qualitative analysis of endothelial cells of donor cornea before & after penetrating keratoplasty in different pathological conditions

    Directory of Open Access Journals (Sweden)

    Aruna K.R. Gupta

    2016-01-01

    Full Text Available Background & objectives: Endothelial cells of the donor cornea are known to be affected quantitatively and qualitatively in different pathological conditions after penetrating keratoplasty (PK and this has direct effect on the clarity of vision obtained after PK. This study was undertaken to analyze the qualitative and quantitative changes in donor endothelial cells before and after PK in different pathological conditions. Methods: A prospective investigational analysis of 100 consecutive donor corneas used for penetrating keratoplasty between June 2006 and June 2008, was conducted. The patients were evaluated on the first day, at the end of first week, first month, third and six months and one year. Results: A decrease was observed in endothelial cell count in all pathological conditions. After one year of follow up the loss was 33.1 per cent in corneal opacity, 45.9 per cent in acute infective keratitis (AIK, 58.5 per cent in regrafts, 28.5 per cent in pseudophakic bullous keratopathy (PBK, 37 per cent in descemetocele, 27 per cent in keratoconus and 35.5 per cent in aphakic bullous keratopathy (ABK cases. Interpretation & conclusions: The endothelial cell loss was highest in regraft cases which was significant (P<0.05, while the least endothelial cell loss was seen in keratoconus cases. The cell loss was associated with increase in coefficient of variation (CV, i.e. polymegathism and pleomorphism. Inspite of this polymegathism and pleomorphism, the clarity of the graft was maintained.

  13. Relationship between endothelial cell loss and microcoaxial phacoemulsification parameters in noncomplicated cataract surgery

    Directory of Open Access Journals (Sweden)

    Soliman Mahdy MAE

    2012-03-01

    Full Text Available Mohamed AE Soliman Mahdy1,2, Mohamed Z Eid1, Mahmoud Abdel-Badei Mohammed3, Amr Hafez4,5, Jagdish Bhatia21Ophthalmic Department, Al-Hussein University Hospital, Al-Azhar University, Cairo, Egypt; 2Ophthalmic Department, Rustaq Hospital, Rustaq, Sultanate of Oman; 3Research Institute of Ophthalmology, Cairo, Egypt; 4Magrabi Eye and Ear Center, Muscat, Sultanate of Oman; 5Ophthalmic Department, Al-Azhar University Hospital, Assuit, EgyptPurpose: To assess the relationship between postoperative endothelial cell loss and microcoaxial phaco parameters using Ozil IP (Alcon Laboratories, Inc, Fort Worth, TX in noncomplicated cataract surgery.Methods: In this prospective observational study, 120 consecutive cases of cataract patients with different grades of nuclear hardness underwent microcoaxial phacoemulsification through a 2.2-mm clear corneal incision. An Alcon Infinity Vision System with Ozil IP (Alcon Laboratories was used with an Ozil torsional handpiece and a Kelman-style 45° phacoemulsification tip. Patients underwent preoperative and postoperative central endothelial cell counts.Results: The study included 120 cases of age-related cataract whose mean age (standard deviation [SD] was 59.68 years (9.47. There was a highly statistically significant endothelial cell loss (P < 0.001. The endothelial cell loss ranged 11–1149 cells/mm2 with a median (interquartile range of 386 cells/mm2 (184.5–686 cells/mm2. The percentage of postoperative ECLoss% ranged from 0.48% to 47.8% with a median (interquartile range of 15.4% (7.2% to 26.8%. A significant positive correlation was found between the ECLoss% and different phaco parameters. The Spearman’s rank-order correlation coefficient values, rho, (ρ were as follows: CDE (ρ = 0.425, aspiration time (ρ = 0.176, and volume (ρ = 0.278. Also, ECLoss% was significantly correlated with the grade of nuclear opalescence (Kendall’s tau τ = 0.42.Conclusion: Microcoaxial phacoemulsification was efficient

  14. Modulation of endothelial cell phenotype by physical activity: impact on obesity-related endothelial dysfunction.

    Science.gov (United States)

    Bender, Shawn B; Laughlin, M Harold

    2015-07-01

    Increased levels of physical activity are associated with reduced cardiovascular disease (CVD) risk and mortality in obesity and diabetes. Available evidence suggests that local factors, including local hemodynamics, account for a significant portion of this CVD protection, and numerous studies have interrogated the therapeutic benefit of physical activity/exercise training in CVD. Less well established is whether basal differences in endothelial cell phenotype between/among vasculatures related to muscle recruitment patterns during activity may account for reports of nonuniform development of endothelial dysfunction in obesity. This is the focus of this review. We highlight recent work exploring the vulnerability of two distinct vasculatures with established differences in endothelial cell phenotype. Specifically, based largely on dramatic differences in underlying hemodynamics, arteries perfusing soleus muscle (slow-twitch muscle fibers) and those perfusing gastrocnemius muscle (fast-twitch muscle fibers) in the rat exhibit an exercise training-like versus an untrained endothelial cell phenotype, respectively. In the context of obesity, therefore, arteries to soleus muscle exhibit protection from endothelial dysfunction compared with vulnerable arteries to gastrocnemius muscle. This disparate vulnerability is consistent with numerous animal and human studies, demonstrating increased skeletal muscle blood flow heterogeneity in obesity coincident with reduced muscle function and exercise intolerance. Mechanistically, we highlight emerging areas of inquiry exploring novel aspects of hemodynamic-sensitive signaling in endothelial cells and the time course of physical activity-associated endothelial adaptations. Lastly, further exploration needs to consider the impact of endothelial heterogeneity on the development of endothelial dysfunction because endothelial dysfunction independently predicts CVD events. Copyright © 2015 the American Physiological Society.

  15. Hypocellular scar formation or aberrant fibrosis induced by an intrastromal corneal ring: a case report

    Directory of Open Access Journals (Sweden)

    Ramkumar Hema L

    2011-08-01

    Full Text Available Abstract Introduction Intrastromal corneal rings or segments are approved for the treatment of myopia and astigmatism associated with keratoconus. We describe a clinicopathological case of intrastromal corneal rings. For the first time, the molecular pathological findings of intrastromal corneal rings in the cornea are illustrated. Case presentation A 47-year-old African-American man with a history of keratoconus and failure in using a Rigid Gas Permeable contact lens received an intrastromal corneal ring implant in his left eye. Due to complications, penetrating keratoplasty was performed. The intrastromal corneal ring channels were surrounded by a dense acellular (channel haze and/or hypocellular (acidophilic densification collagen scar and slightly edematous keratocytes. Mild macrophage infiltration was found near the inner aspect of the intrastromal corneal rings. Molecular analyses of the microdissected cells surrounding the intrastromal corneal ring channels and central corneal stroma revealed 10 times lower relative expression of IP-10/CXCL10 mRNA and two times higher CCL5 mRNA in the cells surrounding the intrastromal corneal ring, as compared to the central corneal stroma. IP-10/CXCL10 is a fibrotic and angiostatic chemokine produced by macrophages, endothelial cells and fibroblasts. Conclusion An intrastromal corneal ring implant can induce hypocellular scar formation and mild inflammation, which may result from aberrant release of fibrosis-related chemokines.

  16. Treatment of corneal squamous cell carcinoma using topical 1% 5-fluorouracil as monotherapy.

    Science.gov (United States)

    Dorbandt, Daniel M; Driskell, Elizabeth A; Hamor, Ralph E

    2016-05-01

    The purpose of this report is to discuss the use of topical 1% 5-fluorouracil as a sole therapy for canine corneal squamous cell carcinoma (SCC). A 12-year-old castrated male pug was evaluated for a well-demarcated, central, 3 mm in diameter, pale pink, raised, right corneal mass. An incisional biopsy was obtained using a #64 beaver blade after topical anesthesia and without sedation. A definitive diagnosis of corneal SCC was obtained after histopathologic evaluation of the biopsy. Topical 1% 5-fluorouracil ointment was applied to the right eye four times daily for 2 weeks followed by no treatment for 2 weeks, then treatment again twice daily for 2 weeks. The cornea remained free of recurrence 10 months after cessation of treatment. In dogs affected with corneal SCC, topical 1% 5-fluorouracil monotherapy may be a viable and cost-effective treatment option with minimal side effects. This chemotherapy agent may also have an effect on corneal pigmentation. Chronic cyclosporine therapy did not contribute to the pathogenesis of corneal SCC in the case described.

  17. Development of Endothelial-Specific Single Inducible Lentiviral Vectors for Genetic Engineering of Endothelial Progenitor Cells.

    Science.gov (United States)

    Yang, Guanghua; Kramer, M Gabriela; Fernandez-Ruiz, Veronica; Kawa, Milosz P; Huang, Xin; Liu, Zhongmin; Prieto, Jesus; Qian, Cheng

    2015-11-27

    Endothelial progenitor cells (EPC) are able to migrate to tumor vasculature. These cells, if genetically modified, can be used as vehicles to deliver toxic material to, or express anticancer proteins in tumor. To test this hypothesis, we developed several single, endothelial-specific, and doxycycline-inducible self-inactivating (SIN) lentiviral vectors. Two distinct expression cassettes were inserted into a SIN-vector: one controlled by an endothelial lineage-specific, murine vascular endothelial cadherin (mVEcad) promoter for the expression of a transactivator, rtTA2S-M2; and the other driven by an inducible promoter, TREalb, for a firefly luciferase reporter gene. We compared the expression levels of luciferase in different vector constructs, containing either the same or opposite orientation with respect to the vector sequence. The results showed that the vector with these two expression cassettes placed in opposite directions was optimal, characterized by a robust induction of the transgene expression (17.7- to 73-fold) in the presence of doxycycline in several endothelial cell lines, but without leakiness when uninduced. In conclusion, an endothelial lineage-specific single inducible SIN lentiviral vector has been developed. Such a lentiviral vector can be used to endow endothelial progenitor cells with anti-tumor properties.

  18. Endothelial progenitor cells and integrins: adhesive needs

    Directory of Open Access Journals (Sweden)

    Caiado Francisco

    2012-03-01

    Full Text Available Abstract In the last decade there have been multiple studies concerning the contribution of endothelial progenitor cells (EPCs to new vessel formation in different physiological and pathological settings. The process by which EPCs contribute to new vessel formation in adults is termed postnatal vasculogenesis and occurs via four inter-related steps. They must respond to chemoattractant signals and mobilize from the bone marrow to the peripheral blood; home in on sites of new vessel formation; invade and migrate at the same sites; and differentiate into mature endothelial cells (ECs and/or regulate pre-existing ECs via paracrine or juxtacrine signals. During these four steps, EPCs interact with different physiological compartments, namely bone marrow, peripheral blood, blood vessels and homing tissues. The success of each step depends on the ability of EPCs to interact, adapt and respond to multiple molecular cues. The present review summarizes the interactions between integrins expressed by EPCs and their ligands: extracellular matrix components and cell surface proteins present at sites of postnatal vasculogenesis. The data summarized here indicate that integrins represent a major molecular determinant of EPC function, with different integrin subunits regulating different steps of EPC biology. Specifically, integrin α4β1 is a key regulator of EPC retention and/or mobilization from the bone marrow, while integrins α5β1, α6β1, αvβ3 and αvβ5 are major determinants of EPC homing, invasion, differentiation and paracrine factor production. β2 integrins are the major regulators of EPC transendothelial migration. The relevance of integrins in EPC biology is also demonstrated by many studies that use extracellular matrix-based scaffolds as a clinical tool to improve the vasculogenic functions of EPCs. We propose that targeted and tissue-specific manipulation of EPC integrin-mediated interactions may be crucial to further improve the usage of

  19. Endothelial protein C receptor in renal tubular epithelial cells and ...

    African Journals Online (AJOL)

    Jane

    2011-07-20

    Jul 20, 2011 ... placenta, heart, liver and lung endothelial cell. However, there ... The effects of some reagents (high glucose, tumor necrosis factor–α and interleukin-1β) were measured by .... functional domains, including N terminal signal peptide ..... endothelial cell protein C receptor (EPCR) 23bp insert in patients with.

  20. Increased endothelial cell-leukocyte interaction in murine schistosomiasis: possible priming of endothelial cells by the disease.

    Directory of Open Access Journals (Sweden)

    Suellen D S Oliveira

    Full Text Available BACKGROUND AND AIMS: Schistosomiasis is an intravascular parasitic disease associated with inflammation. Endothelial cells control leukocyte transmigration and vascular permeability being modulated by pro-inflammatory mediators. Recent data have shown that endothelial cells primed in vivo in the course of a disease keep the information in culture. Herein, we evaluated the impact of schistosomiasis on endothelial cell-regulated events in vivo and in vitro. METHODOLOGY AND PRINCIPAL FINDINGS: The experimental groups consisted of Schistosoma mansoni-infected and age-matched control mice. In vivo infection caused a marked influx of leukocytes and an increased protein leakage in the peritoneal cavity, characterizing an inflamed vascular and cellular profile. In vitro leukocyte-mesenteric endothelial cell adhesion was higher in cultured cells from infected mice as compared to controls, either in the basal condition or after treatment with the pro-inflammatory cytokine tumor necrosis factor (TNF. Nitric oxide (NO donation reduced leukocyte adhesion to endothelial cells from control and infected groups; however, in the later group the effect was more pronounced, probably due to a reduced NO production. Inhibition of control endothelial NO synthase (eNOS increased leukocyte adhesion to a level similar to the one observed in the infected group. Besides, the adhesion of control leukocytes to endothelial cells from infected animals is similar to the result of infected animals, confirming that schistosomiasis alters endothelial cells function. Furthermore, NO production as well as the expression of eNOS were reduced in cultured endothelial cells from infected animals. On the other hand, the expression of its repressor protein, namely caveolin-1, was similar in both control and infected groups. CONCLUSION/SIGNIFICANCE: Schistosomiasis increases vascular permeability and endothelial cell-leukocyte interaction in vivo and in vitro. These effects are partially

  1. Benzalkonium chloride suppresses rabbit corneal endothelium intercellular gap junction communication.

    Directory of Open Access Journals (Sweden)

    Zhenhao Zhang

    Full Text Available Gap junction intercellular communication (GJIC plays a critical role in the maintenance of corneal endothelium homeostasis. We determined if benzalkonium chloride (BAK alters GJIC activity in the rabbit corneal endothelium since it is commonly used as a drug preservative in ocular eyedrop preparations even though it can have cytotoxic effects.Thirty-six adult New Zealand albino rabbits were randomly divided into three groups. BAK at 0.01%, 0.05%, and 0.1% was applied twice daily to one eye of each of the rabbits in one of the three groups for seven days. The contralateral untreated eyes were used as controls. Corneal endothelial morphological features were observed by in vivo confocal microscopy (IVCM. Immunofluorescent staining resolved changes in gap junction integrity and localization. Western blot analysis and RT-PCR evaluated changes in levels of connexin43 (Cx43 and tight junction zonula occludens-1 (ZO-1 gene and protein expression, respectively. Cx43 and ZO-1 physical interaction was detected by immunoprecipitation (IP. Primary rabbit corneal endothelial cells were cultured in Dulbecco's Modified Eagle Medium (DMEM containing BAK for 24 hours. The scrape-loading dye transfer technique (SLDT was used to assess GJIC activity.Topical administration of BAK (0.05%, 0.1% dose dependently disrupted corneal endothelial cell morphology, altered Cx43 and ZO-1 distribution and reduced Cx43 expression. BAK also markedly induced increases in Cx43 phosphorylation status concomitant with decreases in the Cx43-ZO-1 protein-protein interaction. These changes were associated with marked declines in GJIC activity.The dose dependent declines in rabbit corneal endothelial GJIC activity induced by BAK are associated with less Cx43-ZO-1 interaction possibly arising from increases in Cx43 phosphorylation and declines in its protein expression. These novel changes provide additional evidence that BAK containing eyedrop preparations should be used with caution to

  2. Experimental Study of Plasmid TGF-β1 DNA Gene Transfer with Lipofectamine into Rabbit Corneal Epithelial Cells In Vitro

    Institute of Scientific and Technical Information of China (English)

    黄琼; 胡燕华; 姜发纲; 陈宏

    2002-01-01

    To investigate whether the TGF-β1 plasmid DNA carried by lipofectamine could be introduced into cultured rabbit corneal epithelial cells, specific expression of the plasmid pMAM TGF-β1in the cultured corneal epithelial cells was studied. Two days after 12 h of transfection of pMAMTGF-β1 mediated by lipofectamine into the cultured corneal epithelial cells, the TGF-β1 protein expression specific for pMAMTGF-β1 in the cells was detected by means of immunohistochemical staining and the positive rate was 23. 37 %. The results suggested that foreign plasmid DNA could be effectively delivered into cultured rabbit corneal epithelial cells by means of lipofectamine, and this will provide a promising method of studying TGF-β1 on the mechanism of physiology and pathology concerned with corneal epithelial cells.

  3. Efficacy of cultivated corneal epithelial stem cells for ocular surface reconstruction

    Directory of Open Access Journals (Sweden)

    Prabhasawat P

    2012-09-01

    Full Text Available Pinnita Prabhasawat,1 Pattama Ekpo,2 Mongkol Uiprasertkul,3 Suksri Chotikavanich,1 Nattaporn Tesavibul11Department of Ophthalmology, 2Department of Immunology, 3Department of Pathology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, ThailandPurpose: To investigate the clinical outcomes of cultivated corneal limbal epithelial transplantation (CLET using human amniotic membrane for corneal limbal stem-cell deficiency.Methods: Prospective, noncomparative case series. Eighteen patients (19 eyes with severe ocular surface diseases were chosen to undergo CLET using human amniotic membrane. Twelve eyes received auto-CLET, and seven eyes received allo-CLET. Clinical outcomes of corneal surface epithelialization, conjunctivalization, inflammation, visual acuity, graft status, and complications were observed.Results: Corneal epithelium cultivated on amniotic membrane (two to four layers was positive for molecular markers p63, ABCG2, CK3, and CK12. The mean patient age was 44.7 ± 15.2 years. A successful clinical outcome, defined as corneal epithelialization without central conjunctivalization or severe inflammation, was obtained in 14 (73.7% of 19 eyes (mean follow-up 26.1 ± 13.5 months; range 6–47. A histopathologic success, defined as absence of goblet cells at the central cornea, was achieved in 12 (63.2% eyes. Clinical failures occurred in five (26.3% of 19 eyes, and histopathologic failures occurred in seven (36.8% of 19 eyes. Survival analysis at 1 year showed that the clinical success rate was 77.9% and the pathological success rate was 72.3%. Fourteen of 19 (73.7% eyes had visual acuity improvements after CLET. Six cases underwent penetrating keratoplasty; five of these grafts remained clear after 20.4 ± 6.9 months (range, 12–31 of follow-up. Complications included infectious keratitis (three cases and recurrent symblepharon (one case. All complicated cases had lid abnormalities. Factors affecting the final clinical

  4. Aligned Fibrous Scaffold Induced Aligned Growth of Corneal Stroma Cells in vitro Culture

    Institute of Scientific and Technical Information of China (English)

    GAO Yan; YAN Jing; CUI Xue-jun; WANG Hong-yan; WANG Qing

    2012-01-01

    To investigate the contribution of fibre arrangement to guiding the aligned growth of corneal stroma cells,aligned and randomly oriented fibrous scaffolds of gelatin and poly-L-lactic acid(PLLA) were fabricated by electrospinning.A comparative study of two different systems with corneal stroma cells on randomly organized and aligned fibres were conducted.The efficiency of the scaffolds for inducing the aligned growth of cells was assessed by morphological observation and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT) assay.Results show that the cells cultured on both randomly oriented and aligned scaffolds maintained normal morphology and well spreading as well as long term proliferation.Importantly,corneal stroma cells grew high orderly on the aligned scaffold,while the cells grew disordered on the randomly oriented scaffold.Moreover,the cells exhibited higher viability in aligned scaffold than that in randomly oriented scaffold.These results indcate that electrospinng to prepare aligned fibrous scaffolds has provided an effective approach to the aligned growth of corneal stroma cells in vitro.Our findings that fiber arrangement plays a crucial role in guiding the aligned growth of cells may be helpful to the development of better biomaterials for tissue engineered cornea.

  5. Nanoscale topography-induced modulation of fundamental cell behaviors of rabbit corneal keratocytes, fibroblasts, and myofibroblasts.

    Science.gov (United States)

    Pot, Simon A; Liliensiek, Sara J; Myrna, Kathern E; Bentley, Ellison; Jester, James V; Nealey, Paul F; Murphy, Christopher J

    2010-03-01

    Keratocyte-to-myofibroblast differentiation is a key factor in corneal wound healing. The purpose of this study was to determine the influence of environmental nanoscale topography on keratocyte, fibroblast, and myofibroblast cell behavior. Primary rabbit corneal keratocytes, fibroblasts, and myofibroblasts were seeded onto planar polyurethane surfaces with six patterned areas, composed of anisotropically ordered grooves and ridges with a 400-, 800-, 1200-, 1600-, 2000-, and 4000-nm pitch (pitch = groove + ridge width). After 24 hours cells were fixed, stained, imaged, and analyzed for cell shape and orientation. For migration studies, cells on each patterned surface were imaged every 10 minutes for 12 hours, and individual cell trajectories and migration rates were calculated. Keratocytes, fibroblasts, and myofibroblasts aligned and elongated to pitch sizes larger than 1000 nm. A lower limit to the topographic feature sizes that the cells responded to was identified for all three phenotypes, with a transition zone around the 800- to 1200-nm pitch size. Fibroblasts and myofibroblasts migrated parallel to surface ridges larger than 1000 nm but lacked directional guidance on submicron and nanoscale topographic features and on planar surfaces. Keratocytes remained essentially immobile. Corneal stromal cells elongated, aligned, and migrated, differentially guided by substratum topographic features. All cell types failed to respond to topographic features approximating the dimensions of individual stromal fibers. These findings contribute to our understanding of corneal stromal cell biology in health and disease and their interaction with biomaterials and their native extracellular matrix.

  6. A Human Corneal Epithelial Cell Line Model for Limbal Stem Cell Biology and Limbal Immunobiology.

    Science.gov (United States)

    Shaharuddin, Bakiah; Ahmad, Sajjad; Md Latar, Nani; Ali, Simi; Meeson, Annette

    2016-10-14

    : Limbal stem cell (LSC) deficiency is a visually debilitating condition caused by abnormal maintenance of LSCs. It is treated by transplantation of donor-derived limbal epithelial cells (LECs), the success of which depends on the presence and quality of LSCs within the transplant. Understanding the immunobiological responses of these cells within the transplants could improve cell engraftment and survival. However, human corneal rings used as a source of LSCs are not always readily available for research purposes. As an alternative, we hypothesized that a human telomerase-immortalized corneal epithelial cell (HTCEC) line could be used as a model for studying LSC immunobiology. HTCEC constitutively expressed human leukocyte antigen (HLA) class I but not class II molecules. However, when stimulated by interferon-γ, HTCECs then expressed HLA class II antigens. Some HTCECs were also migratory in response to CXCL12 and expressed stem cell markers, Nanog, Oct4, and Sox2. In addition because both HTCECs and LECs contain side population (SP) cells, which are an enriched LSC population, we used these SP cells to show that some HTCEC SP cells coexpressed ABCG2 and ABCB5. HTCEC SP and non-side population (NSP) cells also expressed CXCR4, but the SP cells expressed higher levels. Both were capable of colony formation, but the NSP colonies were smaller and contained fewer cells. In addition, HTCECs expressed ΔNp63α. These results suggest the HTCEC line is a useful model for further understanding LSC biology by using an in vitro approach without reliance on a supply of human tissue. Limbal stem cell deficiency is a painful eye condition caused by abnormal maintenance of limbal stem cells. It is treated by transplantation of limbal epithelial cells derived from human tissue. The success of this treatment depends of the quality of the cells transplanted; however, some transplants fail. Understanding more about the immunobiology of these cells within the transplants could

  7. In vitro ultraviolet–induced damage in human corneal, lens, and retinal pigment epithelial cells

    OpenAIRE

    Youn, Hyun-Yi; McCanna, David J.; Sivak, Jacob G.; Jones, Lyndon W.

    2011-01-01

    Purpose The purpose was to develop suitable in vitro methods to detect ocular epithelial cell damage when exposed to UV radiation, in an effort to evaluate UV-absorbing ophthalmic biomaterials. Methods Human corneal epithelial cells (HCEC), lens epithelial cells (HLEC), and retinal pigment epithelial cells (ARPE-19) were cultured and Ultraviolet A/Ultraviolet B (UVA/UVB) blocking filters and UVB-only blocking filters were placed between the cells and a UV light source. Cells were irradiated w...

  8. The Toxicity of Nonsteroidal Anti-inflammatory Eye Drops against Human Corneal Epithelial Cells in Vitro.

    Science.gov (United States)

    Lee, Jong Soo; Kim, Young Hi; Park, Young Min

    2015-12-01

    This study investigated the toxicity of commercial non-steroid anti-inflammatory drug (NSAID) eye solutions against corneal epithelial cells in vitro. The biologic effects of 1/100-, 1/50-, and 1/10-diluted bromfenac sodium, pranoprofen, diclofenac sodium, and the fluorometholone on corneal epithelial cells were evaluated after 1-, 4-, 12-, and 24-hr of exposure compared to corneal epithelial cell treated with balanced salt solution as control. Cellular metabolic activity, cellular damage, and morphology were assessed. Corneal epithelial cell migration was quantified by the scratch-wound assay. Compared to bromfenac and pranoprofen, the cellular metabolic activity of diclofenac and fluorometholone significantly decreased after 12-hr exposure, which was maintained for 24-hr compared to control. Especially, at 1/10-diluted eye solution for 24-hr exposure, the LDH titers of fluorometholone and diclofenac sodium markedly increased more than those of bromfenac and pranoprofen. In diclofenac sodium, the Na(+) concentration was lower and amount of preservatives was higher than other NSAIDs eye solutions tested. However, the K(+) and Cl(-) concentration, pH, and osmolarity were similar for all NSAIDs eye solutions. Bromfenac and pranoprofen significantly promoted cell migration, and restored wound gap after 48-hr exposure, compared with that of diclofenac or fluorometholone. At 1/50-diluted eye solution for 48-hr exposure, the corneal epithelial cellular morphology of diclofenac and fluorometholone induced more damage than that of bromfenac or pranoprofen. Overall, the corneal epithelial cells in bromfenac and pranoprofen NSAID eye solutions are less damaged compared to those in diclofenac, included fluorometholone as steroid eye solution.

  9. Adhesion and metabolic activity of human corneal cells on PCL based nanofiber matrices.

    Science.gov (United States)

    Stafiej, Piotr; Küng, Florian; Thieme, Daniel; Czugala, Marta; Kruse, Friedrich E; Schubert, Dirk W; Fuchsluger, Thomas A

    2017-02-01

    In this work, polycaprolactone (PCL) was used as a basic polymer for electrospinning of random and aligned nanofiber matrices. Our aim was to develop a biocompatible substrate for ophthalmological application to improve wound closure in defects of the cornea as replacement for human amniotic membrane. We investigated whether blending the hydrophobic PCL with poly (glycerol sebacate) (PGS) or chitosan (CHI) improves the biocompatibility of the matrices for cell expansion. Human corneal epithelial cells (HCEp) and human corneal keratocytes (HCK) were used for in vitro biocompatibility studies. After optimization of the electrospinning parameters for all blends, scanning electron microscopy (SEM), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), and water contact angle were used to characterize the different matrices. Fluorescence staining of the F-actin cytoskeleton of the cells was performed to analyze the adherence of the cells to the different matrices. Metabolic activity of the cells was measured by cell counting kit-8 (CCK-8) for 20days to compare the biocompatibility of the materials. Our results show the feasibility of producing uniform nanofiber matrices with and without orientation for the used blends. All materials support adherence and proliferation of human corneal cell lines with oriented growth on aligned matrices. Although hydrophobicity of the materials was lowered by blending PCL, no increase in biocompatibility or proliferation, as was expected, could be measured. All tested matrices supported the expansion of human corneal cells, confirming their potential as substrates for biomedical applications.

  10. ICAM-1 is necessary for epithelial recruitment of gammadelta T cells and efficient corneal wound healing.

    Science.gov (United States)

    Wound healing and inflammation are both significantly reduced in mice that lack gammadelta T cells. Here, the role of epithelial intercellular adhesion molecule-1 (ICAM-1) in gammadelta T cell migration in corneal wound healing was assessed. Wild-type mice had an approximate fivefold increase in epi...

  11. NK cells modulate the inflammatory response to corneal epithelial abrasion and thereby support wound healing

    Science.gov (United States)

    Natural killer cells are lymphocytes of the innate immune system that have crucial cytotoxic and regulatory roles in adaptive immunity and inflammation. Herein, we consider a role for these cells in corneal wound healing. After a 2-mm central epithelial abrasion of the mouse cornea, a subset of clas...

  12. Silencing of directional migration in roundabout4 knockdown endothelial cells

    Directory of Open Access Journals (Sweden)

    Roberts David D

    2008-11-01

    Full Text Available Abstract Background Roundabouts are axon guidance molecules that have recently been identified to play a role in vascular guidance as well. In this study, we have investigated gene knockdown analysis of endothelial Robos, in particular roundabout 4 (robo4, the predominant Robo in endothelial cells using small interfering RNA technology in vitro. Results Robo1 and Robo4 knockdown cells display distinct activity in endothelial cell migration assay. The knockdown of robo4 abrogated the chemotactic response of endothelial cells to serum but enhanced a chemokinetic response to Slit2, while robo1 knockdown cells do not display chemotactic response to serum or VEGF. Robo4 knockdown endothelial cells unexpectedly show up regulation of Rho GTPases. Zebrafish Robo4 rescues both Rho GTPase homeostasis and serum reduced chemotaxis in robo4 knockdown cells. Robo1 and Robo4 interact and share molecules such as Slit2, Mena and Vilse, a Cdc42-GAP. In addition, this study mechanistically implicates IRSp53 in the signaling nexus between activated Cdc42 and Mena, both of which have previously been shown to be involved with Robo4 signaling in endothelial cells. Conclusion This study identifies specific components of the Robo signaling apparatus that work together to guide directional migration of endothelial cells.

  13. Caspases and p38 MAPK regulate endothelial cell adhesiveness for mesenchymal stem cells.

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    Irina A Potapova

    Full Text Available Mesenchymal stem cells natively circulating or delivered into the blood stream home to sites of injury. The mechanism of mesenchymal stem cell homing to sites of injury is poorly understood. We have shown that the development of apoptosis in endothelial cells stimulates endothelial cell adhesiveness for mesenchymal stem cells. Adhesion of mesenchymal stem cells to apoptotic endothelial cells depends on the activation of endothelial caspases and p38 MAPK. Activation of p38 MAPK in endothelial cells has a primary effect while the activation of caspases potentiates the mesenchymal stem cell adhesion. Overall, our study of the mesenchymal stem cell interaction with endothelial cells indicates that mesenchymal stem cells recognize and specifically adhere to distressed/apoptotic endothelial cells.

  14. Limbal Stromal Tissue Specific Stem Cells and Their Differentiation Potential to Corneal Epithelial Cells.

    Science.gov (United States)

    Katikireddy, Kishore Reddy; Jurkunas, Ula V

    2016-01-01

    From the derivation of the first human embryonic stem (hES) cell line to the development of induced pluripotent stem (iPS) cells; it has become evident that tissue specific stem cells are able to differentiate into a specific somatic cell types. The understanding of key processes such as the signaling pathways and the role of the microenvironment in epidermal/epithelial development has provided important clues for the derivation of specific epithelial cell types.Various differentiation protocols/methods were used to attain specific epithelial cell types. Here, we describe in detail the procedure to follow for isolation of tissue specific stem cells, mimicking their microenvironment to attain stem cell characteristics, and their potential differentiation to corneal epithelial cells.

  15. Angiogenic potential of endothelial progenitor cells and embryonic stem cells

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    Rae Peter C

    2011-05-01

    Full Text Available Abstract Background Endothelial progenitor cells (EPCs are implicated in a range of pathological conditions, suggesting a natural therapeutic role for EPCs in angiogenesis. However, current angiogenic therapies involving EPC transplantation are inefficient due to rejection of donor EPCs. One solution is to derive an expanded population of EPCs from stem cells in vitro, to be re-introduced as a therapeutic transplant. To demonstrate the therapeutic potential of EPCs we performed in vitro transplantation of EPCs into endothelial cell (EC tubules using a gel-based tubule formation assay. We also described the production of highly angiogenic EPC-comparable cells from pluripotent embryonic stem cells (ESCs by direct differentiation using EC-conditioned medium (ECCM. Results The effect on tubule complexity and longevity varied with transplantation quantity: significant effects were observed when tubules were transplanted with a quantity of EPCs equivalent to 50% of the number of ECs originally seeded on to the assay gel but not with 10% EPC transplantation. Gene expression of the endothelial markers VEGFR2, VE-cadherin and CD31, determined by qPCR, also changed dynamically during transplantation. ECCM-treated ESC-derived progenitor cells exhibited angiogenic potential, demonstrated by in vitro tubule formation, and endothelial-specific gene expression equivalent to natural EPCs. Conclusions We concluded the effect of EPCs is cumulative and beneficial, relying on upregulation of the angiogenic activity of transplanted cells combined with an increase in proliferative cell number to produce significant effects upon transplantation. Furthermore, EPCs derived from ESCs may be developed for use as a rapidly-expandable alternative for angiogenic transplantation therapy.

  16. p53 protein expression in corneal squamous cell carcinomas of dogs

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    Lucas Bahdour Cossi

    2015-06-01

    Full Text Available Ocular tumors play an increasing concern in veterinary ophthalmology. Corneal squamous cell carcinoma is unfrequent in dogs, and by this way it has little studies. Studies that investigated the carcinogenesis mechanisms wich could help to the development of ocular squamous cell carcinoma (SCC in dog are rare. The aim of this work was to identify by immunohistochemical techniques, the p53 protein expression in the spontaneous dog corneal SCC. For this work, were used five cases of corneal SCC and one case of actinic keratitis. The sections were obtained from paraffin-wax blocks and submitted to histopathological and immunohistochemical analysis. All the six samples showed immunolabeling to cytokeratin and p53 protein. These results support the conclusions that the immunoreactivity of p53 protein by immunohistochemistry is present in canine corneal SCC suppporting its role in carcinogenesis of this tumor, but not provides prognostic indicators in cases of SCC corneal in dog; and can be a association of exposure to solar radiation with the possible mutation of the TP53 gene.

  17. CTR1 silencing inhibits angiogenesis by limiting copper entry into endothelial cells.

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    Gomathy Narayanan

    Full Text Available Increased levels of intracellular copper stimulate angiogenesis in human umbilical vein endothelial cells (HUVECs. Copper transporter 1 (CTR1 is a copper importer present in the cell membrane and plays a major role in copper transport. In this study, three siRNAs targeting CTR1 mRNA were designed and screened for gene silencing. HUVECs when exposed to 100 µM copper showed 3 fold increased proliferation, migration by 1.8-fold and tube formation by 1.8-fold. One of the designed CTR1 siRNA (si 1 at 10 nM concentration decreased proliferation by 2.5-fold, migration by 4-fold and tube formation by 2.8-fold. Rabbit corneal packet assay also showed considerable decrease in matrigel induced blood vessel formation by si 1 when compared to untreated control. The designed si 1 when topically applied inhibited angiogenesis. This can be further developed for therapeutic application.

  18. Variations in mass transfer to single endothelial cells.

    Science.gov (United States)

    Van Doormaal, Mark A; Zhang, Ji; Wada, Shigeo; Shaw, James E; Won, Doyon; Cybulsky, Myron I; Yip, Chris M; Ethier, C Ross

    2009-06-01

    Mass transfer between flowing blood and arterial mural cells (including vascular endothelial cells) may play an important role in atherogenesis. Endothelial cells are known to have an apical surface topography that is not flat, and hence mass transfer patterns to individual endothelial cells are likely affected by the local cellular topography. The purpose of this paper is to investigate the relationship between vascular endothelial cell surface topography and cellular level mass transfer. Confluent porcine endothelial monolayers were cultured under both shear and static conditions and atomic force microscopy was used to measure endothelial cell topography. Using finite element methods and the measured cell topography, flow and concentration fields were calculated for a typical, small, blood-borne solute. A relative Sherwood number was defined as the difference between the computed Sherwood number and that predicted by the Leveque solution for mass transfer over a flat surface: this eliminates the effects of axial location on mass transfer efficiency. The average intracellular relative Sherwood number range was found to be dependent on cell height and not dependent on cell elongation due to shear stress in culture. The mass flux to individual cells reached a maximum at the highest point on the endothelial cell surface, typically corresponding to the nucleus of the cell. Therefore, for small receptor-mediated solutes, increased solute uptake efficiency can be achieved by concentrating receptors near the nucleus. The main conclusion of the work is that although the rate of mass transfer varies greatly over an individual cell, the average mass transfer rate to a cell is close to that predicted for a flat cell. In comparison to other hemodynamic factors, the topography of endothelial cells therefore seems to have little effect on mass transfer rates and is likely physiologically insignificant.

  19. Genipin inhibits endothelial exocytosis via nitric oxide in cultured human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Guang-fa WANG; Shao-yu WU; Jin-jun RAO; Lin L(U); Wei XU; Jian-xin PANG; Zhong-qiu LIU; Shu-guang WU; Jia-jie ZHANG

    2009-01-01

    Aim: Exocytosis of endothelial Weibel-Palade bodies, which contain von Willebrand factor (VWF), P-selectin and other modulators, plays an important role in both inflammation and thrombosis. The present study investigates whether genipin,an aglycon of geniposide, inhibits endothelial exocytosis.Methods: Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords and cultured. The concentration of VWF in cell supernatants was measured using an ELISA Kit. P-selectin translocation on the cell surface was analyzed by cell surface ELISA. Cell viability was measured using a Cell Counting Kit-8. Mouse bleeding times were measured by amputating the tail tip. Western blot analysis was used to determine the amount of endothelial nitric oxide synthase (eNOS) and phospho-eNOS present. Nitric oxide (NO) was measured in the cell supernatants as nitrite using an NO Colorimetric Assay.Results: Genipin inhibited thrombin-induced VWF release and P-selectin translocation in HUVECs in a dose- and time-dependent manner. The drug had no cytotoxic effect on the cells at the same doses that were able to inhibit exocytosis. The functional study that demonstrated that genipin inhibited exocytosis in vivo also showed that genipin prolonged the mouse bleeding time. Furthermore, genipin activated eNOS phosphorylation, promoted enzyme activation and increased NO production. L-NAME, an inhibitor of NOS, reversed the inhibitory effects of genipin on endothelial exocytosis.Conclusion: Genipin inhibits endothelial exocytosis in HUVECs. The mechanism by which this compound inhibits exocytosis may be related to its ability to stimulate eNOS activation and NO production. Our findings suggest a novel antiinflammatory mechanism for genipin. This compound may represent a new treatment for inflammation and/or thrombosis in which excess endothelial exocytosis plays a pathophysiological role.

  20. Rebamipide increases the mucin-like glycoprotein production in corneal epithelial cells.

    Science.gov (United States)

    Takeji, Yasuhiro; Urashima, Hiroki; Aoki, Akihiro; Shinohara, Hisashi

    2012-06-01

    Dry eye is a multifactorial disease of tears and the ocular surface due to tear deficiency or excessive tear evaporation. Tear film instability is due to a disturbance in ocular surface mucin leading to a dysfunction of mucin, resulting in dry eye. In this study, we examined the effect of rebamipide, an anti-ulcer agent, on glycoconjugate production, as an indicator of mucin-like glycoprotein in cultured corneal epithelial cells. Further, we investigated the effect of rebamipide on the gene expression of membrane-associated mucins. Confluent cultured human corneal epithelial cells were incubated with rebamipide for 24 h. The glycoconjugate content in the supernatant and the cell extracts was measured by wheat germ agglutinin-enzyme-linked lectin assay combined gel-filtration method. In the experiment on mucin gene expression, cultured human corneal epithelial cells were collected at 0, 3, 6, and 12 h after administration of rebamipide. Real-time quantitative polymerase chain reaction was used to analyze the quantity of MUC1, MUC 4, and MUC16 gene expression. Rebamipide significantly increased the glycoconjugate contents in the supernatant and cell extract. In the mucin gene expression in the cells, rebamipide increased MUC1 and MUC4 gene expression, but did not increase MUC16 gene expression. Rebamipide promoted glycoconjugate, which has a property as a mucin-like glycoprotein, in human corneal epithelial cells. The increased production was mediated by MUC1 and MUC4 gene expression.

  1. Lung endothelial cells strengthen, but brain endothelial cells weaken barrier properties of a human alveolar epithelium cell culture model.

    Science.gov (United States)

    Neuhaus, Winfried; Samwer, Fabian; Kunzmann, Steffen; Muellenbach, Ralf M; Wirth, Michael; Speer, Christian P; Roewer, Norbert; Förster, Carola Y

    2012-11-01

    The blood-air barrier in the lung consists of the alveolar epithelium, the underlying capillary endothelium, their basement membranes and the interstitial space between the cell layers. Little is known about the interactions between the alveolar and the blood compartment. The aim of the present study was to gain first insights into the possible interplay between these two neighbored cell layers. We established an in vitro Transwell model of the alveolar epithelium based on human cell line H441 and investigated the influence of conditioned medium obtained from human lung endothelial cell line HPMEC-ST1.6R on the barrier properties of the H441 layers. As control for tissue specificity H441 layers were exposed to conditioned medium from human brain endothelial cell line hCMEC/D3. Addition of dexamethasone was necessary to obtain stable H441 cell layers. Moreover, dexamethasone increased expression of cell type I markers (caveolin-1, RAGE) and cell type II marker SP-B, whereas decreased the transepithelial electrical resistance (TEER) in a concentration dependent manner. Soluble factors obtained from the lung endothelial cell line increased the barrier significantly proven by TEER values and fluorescein permeability on the functional level and by the differential expression of tight junctional proteins on the molecular level. In contrast to this, soluble factors derived from brain endothelial cells weakened the barrier significantly. In conclusion, soluble factors from lung endothelial cells can strengthen the alveolar epithelium barrier in vitro, which suggests communication between endothelial and epithelial cells regulating the integrity of the blood-air barrier.

  2. Autocrine VEGF isoforms differentially regulate endothelial cell behavior

    Directory of Open Access Journals (Sweden)

    Hideki Yamamoto

    2016-09-01

    Full Text Available Vascular endothelial growth factor A (VEGF is involved in all the essential biology of endothelial cells, from proliferation to vessel function, by mediating intercellular interactions and monolayer integrity. It is expressed as three major alternative spliced variants. In mice, these are VEGF120, VEGF164, and VEGF188, each with different affinities for extracellular matrices and cell surfaces, depending on the inclusion of heparin-binding sites, encoded by exons 6 and 7. To determine the role of each VEGF isoform in endothelial homeostasis, we compared phenotypes of primary endothelial cells isolated from lungs of mice expressing single VEGF isoforms in normoxic and hypoxic conditions. The differential expression and distribution of VEGF isoforms affect endothelial cell functions, such as proliferation, adhesion, migration and integrity, which are dependent on the stability of and affinity to VEGF receptor 2 (VEGFR2. We found a correlation between autocrine VEGF164 and VEGFR2 stability, which is also associated with increased expression of proteins involved in cell adhesion. Endothelial cells expressing only VEGF188, which localizes to extracellular matrices or cell surfaces, presented a mesenchymal morphology and weakened monolayer integrity. Cells expressing only VEGF120 lacked stable VEGFR2 and dysfunctional downstream processes, rendering the cells unviable. Endothelial cells expressing these different isoforms in isolation also had differing rates of apoptosis, proliferation, and signaling via nitric oxide (NO synthesis. These data indicate that autocrine signaling of each VEGF isoform has unique functions on endothelial homeostasis and response to hypoxia, due to both distinct VEGF distribution and VEGFR2 stability, which appears to be, at least partly, affected by differential NO production. This study demonstrates that each autocrine VEGF isoform has a distinct effect on downstream functions, namely VEGFR2-regulated endothelial cell

  3. Targeting Endothelial Cells with Multifunctional GaN/Fe Nanoparticles

    Science.gov (United States)

    Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Andrée, Birgit; Cebotari, Serghei; Boyle, Erin C.; Haverich, Axel; Hilfiker, Andres

    2017-08-01

    In this paper, we report on the interaction of multifunctional nanoparticles with living endothelial cells. The nanoparticles were synthesized using direct growth of gallium nitride on zinc oxide nanoparticles alloyed with iron oxide followed by core decomposition in hydrogen flow at high temperature. Using transmission electron microscopy, we demonstrate that porcine aortic endothelial cells take up GaN-based nanoparticles suspended in the growth medium. The nanoparticles are deposited in vesicles and the endothelial cells show no sign of cellular damage. Intracellular inert nanoparticles are used as guiding elements for controlled transportation or designed spatial distribution of cells in external magnetic fields.

  4. Enterococcus faecalis internalization in human umbilical vein endothelial cells (HUVEC).

    Science.gov (United States)

    Millán, Diana; Chiriboga, Carlos; Patarroyo, Manuel A; Fontanilla, Marta R

    2013-04-01

    Initial Enterococcus faecalis-endothelial cell molecular interactions which lead to enterococci associating in the host endothelial tissue, colonizing it and proliferating there can be assessed using in vitro models. Cultured human umbilical vein endothelial cells (HUVEC) have been used to study other Gram-positive bacteria-cell interactions; however, few studies have been aimed at establishing the relationship of E. faecalis with endothelial cells. The aggregation substance (AS) family of adhesins represents an E. faecalis virulence factor which has been implicated in endocarditis severity and bacterial persistence. The Asc10 protein (a member of this family) promotes bacterium-bacterium aggregation and bacterium-host cell binding. Evaluating Asc10 role in bacterial internalization by cultured enterocytes has shown that this adhesin facilitates E. faecalis endocytosis by HT-29 cells. A few eukaryotic cell structural components, such as cytoskeletal proteins, have been involved in E. faecalis entry into cell-lines; it is thus relevant to determine whether Asc10, as well as microtubules and actin microfilaments, play a role in E. faecalis internalization by cultured endothelial cells. The role of Asc10 and cytoskeleton proteins in E. faecalis ability to enter HUVEC was assessed in the present study, as well as cell apoptosis induction by enterococcal internalization by HUVEC; the data indicated increased cell apoptosis and that cytoskeleton components were partially involved in E. faecalis entry to endothelial cells, thereby suggesting that E. faecalis Asc10 protein would not be a critical factor for bacterial entry to cultured HUVEC.

  5. Mesenchymal Stem/Multipotent Stromal Cells from Human Decidua Basalis Reduce Endothelial Cell Activation.

    Science.gov (United States)

    Alshabibi, Manal A; Al Huqail, Al Joharah; Khatlani, Tanvir; Abomaray, Fawaz M; Alaskar, Ahmed S; Alawad, Abdullah O; Kalionis, Bill; Abumaree, Mohamed Hassan

    2017-09-15

    Recently, we reported the isolation and characterization of mesenchymal stem cells from the decidua basalis of human placenta (DBMSCs). These cells express a unique combination of molecules involved in many important cellular functions, which make them good candidates for cell-based therapies. The endothelium is a highly specialized, metabolically active interface between blood and the underlying tissues. Inflammatory factors stimulate the endothelium to undergo a change to a proinflammatory and procoagulant state (ie, endothelial cell activation). An initial response to endothelial cell activation is monocyte adhesion. Activation typically involves increased proliferation and enhanced expression of adhesion and inflammatory markers by endothelial cells. Sustained endothelial cell activation leads to a type of damage to the body associated with inflammatory diseases, such as atherosclerosis. In this study, we examined the ability of DBMSCs to protect endothelial cells from activation through monocyte adhesion, by modulating endothelial proliferation, migration, adhesion, and inflammatory marker expression. Endothelial cells were cocultured with DBMSCs, monocytes, monocyte-pretreated with DBMSCs and DBMSC-pretreated with monocytes were also evaluated. Monocyte adhesion to endothelial cells was examined following treatment with DBMSCs. Expression of endothelial cell adhesion and inflammatory markers was also analyzed. The interaction between DBMSCs and monocytes reduced endothelial cell proliferation and monocyte adhesion to endothelial cells. In contrast, endothelial cell migration increased in response to DBMSCs and monocytes. Endothelial cell expression of adhesion and inflammatory molecules was reduced by DBMSCs and DBMSC-pretreated with monocytes. The mechanism of reduced endothelial proliferation involved enhanced phosphorylation of the tumor suppressor protein p53. Our study shows for the first time that DBMSCs protect endothelial cells from activation by

  6. The Novel Methods for Analysis of Exosomes Released from Endothelial Cells and Endothelial Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Jinju Wang

    2016-01-01

    Full Text Available Exosomes (EXs are cell-derived vesicles that mediate cell-cell communication and could serve as biomarkers. Here we described novel methods for purification and phenotyping of EXs released from endothelial cells (ECs and endothelial progenitor cells (EPCs by combining microbeads and fluorescence quantum dots (Q-dots® techniques. EXs from the culture medium of ECs and EPCs were isolated and detected with cell-specific antibody conjugated microbeads and second antibody conjugated Q-dots by using nanoparticle tracking analysis (NTA system. The sensitivities of the cell origin markers for ECs (CD105, CD144 and EPCs (CD34, KDR were evaluated. The sensitivity and specificity were determined by using positive and negative markers for EXs (CD63, platelets (CD41, erythrocytes (CD235a, and microvesicles (Annexin V. Moreover, the methods were further validated in particle-free plasma and patient samples. Results showed that anti-CD105/anti-CD144 and anti-CD34/anti-KDR had the highest sensitivity and specificity for isolating and detecting EC-EXs and EPC-EXs, respectively. The methods had the overall recovery rate of over 70% and were able to detect the dynamical changes of circulating EC-EXs and EPC-EXs in acute ischemic stroke. In conclusion, we have developed sensitive and specific microbeads/Q-dots fluorescence NTA methods for EC-EX and EPC-EX isolation and detection, which will facilitate the functional study and biomarker discovery.

  7. Insulin resistance in vascular endothelial cells promotes intestinal tumour formation

    DEFF Research Database (Denmark)

    Wang, X; Häring, M-F; Rathjen, Thomas

    2017-01-01

    in tumour endothelial cells produces an activated, proinflammatory state that promotes tumorigenesis. Improvement of endothelial dysfunction may reduce colorectal cancer risk in patients with obesity and type 2 diabetes.Oncogene advance online publication, 1 May 2017; doi:10.1038/onc.2017.107....

  8. Refractive improvements and safety with topography-guided corneal crosslinking for keratoconus: 1-year results.

    Science.gov (United States)

    Nordström, Maria; Schiller, Maria; Fredriksson, Anneli; Behndig, Anders

    2017-07-01

    To assess the refractive improvements and the corneal endothelial safety of an individualised topography-guided regimen for corneal crosslinking in progressive keratoconus. An open-label prospective randomised clinical trial was performed at the Department of Clinical Sciences, Ophthalmology, Umeå University Hospital, Umeå, Sweden. Thirty-seven patients (50 eyes) with progressive keratoconus planned for corneal crosslinking were included. The patients were randomised to topography-guided crosslinking (photorefractive intrastromal crosslinking (PiXL); n=25) or uniform 9 mm crosslinking (corneal collagen crosslinking (CXL); n=25). Visual acuity, refraction, keratometry (K1, K2 and Kmax) and corneal endothelial morphometry were assessed preoperatively and at 1, 3, 6 and 12 months postoperatively. The PiXL treatment involved an asymmetrical treatment zone centred on the area of maximum corneal steepness with treatment energies ranging from 7.2 to 15.0 J/cm(2); the CXL treatment was a uniform 9 mm 5.4 J/cm(2) pulsed crosslinking. The main outcome measures were changes in refractive errors and corneal endothelial cell density. The spherical refractive errors decreased (pkeratoconus with decreased spherical refractive errors and improved visual acuity, without damage to the corneal endothelium. NCT02514200, Results. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  9. Sodium renders endothelial cells sticky for red blood cells

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    Hans eOberleithner

    2015-06-01

    Full Text Available Negative charges in the glycocalyx of red blood cells (RBC and vascular endothelial cells (EC facilitate frictionless blood flow through blood vessels. Na+ selectively shields these charges controlling surface electronegativity. The question was addressed whether the ambient Na+ concentration controls RBC-EC interaction. Using atomic force microscopy (AFM adhesion forces between RBC and endothelial glycocalyx were quantified. A single RBC, mounted on an AFM cantilever, was brought in physical contact with the endothelial surface and then pulled off. Adhesion forces were quantified (i after enzymatic removal of negative charges in the glycocalyx, (ii under different ambient Na+ and (iii after applying the intracellular aldosterone receptor antagonist spironolactone. Removal of negative surface charges increases RBC-EC interaction forces. A stepwise increase of ambient Na+ from 133 to 140 mM does not affect them. However, beyond 140 mM Na+ adhesion forces increase sharply (10% increase of adhesion force per 1 mM increase of Na+. Spironolactone prevents this response. It is concluded that negative charges reduce adhesion between RBC and EC. Ambient Na+ concentration determines the availability of free negative charges. Na+ concentrations in the low physiological range (below 140 mM allow sufficient amounts of vacant negative charges so that adhesion of RBC to the endothelial surface is small. In contrast, Na+ in the high physiological range (beyond 140 mM saturates the remaining negative surface charges thus increasing adhesion. Aldosterone receptor blockade by spironolactone prevents Na+ induced RBC adhesion to the endothelial glycocalyx. Extrapolation of in vitro experiments to in vivo conditions leads to the hypothesis that high sodium intake is likely to increase the incidence of thrombotic events.

  10. Sodium renders endothelial cells sticky for red blood cells.

    Science.gov (United States)

    Oberleithner, Hans; Wälte, Mike; Kusche-Vihrog, Kristina

    2015-01-01

    Negative charges in the glycocalyx of red blood cells (RBC) and vascular endothelial cells (EC) facilitate frictionless blood flow through blood vessels. Na(+) selectively shields these charges controlling surface electronegativity. The question was addressed whether the ambient Na(+) concentration controls RBC-EC interaction. Using atomic force microscopy (AFM) adhesion forces between RBC and endothelial glycocalyx were quantified. A single RBC, mounted on an AFM cantilever, was brought in physical contact with the endothelial surface and then pulled off. Adhesion forces were quantified (i) after enzymatic removal of negative charges in the glycocalyx, (ii) under different ambient Na(+) and (iii) after applying the intracellular aldosterone receptor antagonist spironolactone. Removal of negative surface charges increases RBC-EC interaction forces. A stepwise increase of ambient Na(+) from 133 to 140 mM does not affect them. However, beyond 140 mM Na(+) adhesion forces increase sharply (10% increase of adhesion force per 1 mM increase of Na(+)). Spironolactone prevents this response. It is concluded that negative charges reduce adhesion between RBC and EC. Ambient Na(+) concentration determines the availability of free negative charges. Na(+) concentrations in the low physiological range (below 140 mM) allow sufficient amounts of vacant negative charges so that adhesion of RBC to the endothelial surface is small. In contrast, Na(+) in the high physiological range (beyond 140 mM) saturates the remaining negative surface charges thus increasing adhesion. Aldosterone receptor blockade by spironolactone prevents Na(+) induced RBC adhesion to the endothelial glycocalyx. Extrapolation of in vitro experiments to in vivo conditions leads to the hypothesis that high sodium intake is likely to increase the incidence of thrombotic events.

  11. Rethinking immunological privilege: implications for corneal and limbal stem cell transplantation.

    Science.gov (United States)

    Williams, K A; Coster, D J

    1997-11-01

    Immunological privilege operates within the normal eye by multiple passive and active mechanisms, including antigen sequestration, maintenance of an immunosuppressive local environment and induction of apoptotic death in infiltrating cells of the immune system. Ocular privilege might have developed to protect the eye from the collateral damage associated with an inflammatory response to invading pathogens. Nevertheless, corneal grafts do undergo irreversible immunological rejection and, furthermore, corneal graft rejection is very similar at a histological level to the rejection processes that operate in vascularized organ grafts. Ocular privilege is thus relative. The question arises as to how corneal grafts are rejected in the face of so many mechanisms designed to prevent immune responses from operating inside the eye--a question that is still essentially unanswered.

  12. Biophysical Cueing and Vascular Endothelial Cell Behavior

    Directory of Open Access Journals (Sweden)

    Joshua A. Wood

    2010-03-01

    Full Text Available Human vascular endothelial cells (VEC line the vessels of the body and are critical for the maintenance of vessel integrity and trafficking of biochemical cues. They are fundamental structural elements and are central to the signaling environment. Alterations in the normal functioning of the VEC population are associated with a number of vascular disorders among which are some of the leading causes of death in both the United States and abroad. VECs attach to their underlying stromal elements through a specialization of the extracellular matrix, the basement membrane. The basement membrane provides signaling cues to the VEC through its chemical constituents, by serving as a reservoir for cytoactive factors and through its intrinsic biophysical properties. This specialized matrix is composed of a topographically rich 3D felt-like network of fibers and pores on the nano (1–100 nm and submicron (100–1,000 nm size scale. The basement membrane provides biophysical cues to the overlying VECs through its intrinsic topography as well as through its local compliance (relative stiffness. These biophysical cues modulate VEC adhesion, migration, proliferation, differentiation, and the cytoskeletal signaling network of the individual cells. This review focuses on the impact of biophysical cues on VEC behaviors and demonstrates the need for their consideration in future vascular studies and the design of improved prosthetics.

  13. Effects of diabetic HDL on endothelial cell function.

    Science.gov (United States)

    He, Dan; Pan, Bing; Ren, Hui; Zheng, Lemin

    2014-01-01

    Type 2 diabetes mellitus (T2DM) is accompanied by dysfunctional high-density lipoprotein (HDL) and this is characterized by alterations in its composition and structure compared with HDL from normal subjects (N-HDL). HDL from diabetic subjects (D-HDL) has a diminished endothelial protective capacity including reducted ability to exert antioxidative activity, stimulate endothelial cell (EC) production of nitric oxide (NO) and endothelium-dependent vasomotion, promote endothelial progenitor cell (EPC)-mediated endothelial repair. In addition, D-HDL promotes EC proliferation, migration and adhesion to the matrix. The present review provides an overview of these effects of diabetic HDL on EC function, as well as the possible changes of D-HDL structure and composition which may be responsible for the diminished endothelial protective capacity of D-HDL.

  14. Effect of pirfenidone on the proliferation of rat corneal stromal cells

    Directory of Open Access Journals (Sweden)

    Jun-Jie Chen

    2015-02-01

    Full Text Available AIM: To investigate the effects of pirfenidone(PFDon the proliferation and transfomring growth factor-β1(TGF-β1expression in vitro culture rat corneal stromal cells. METHODS: Corneal stromal cells from 8 to 10wk SD rats were isolated, cultured and treated with different concentrations of PFD 0mg/mL(control group, 0.15mg/mL(experimental group Ⅰ, 0.3mg/mL(experimental group Ⅱ, 1mg/mL(experimental group Ⅲfor 48h. CCK-8 assay was performed to assess cell proliferation, while immunocytochemistry and Western Blot were used to detect the expression of ki-67 and TGF-β1 expression, respectively. RESULTS: Compared with control group, PFD significantly inhibited the proliferation in a dose-dependent manner(all P1 in a dose-dependent manner(PCONCLUSION: Pirfenidone can significantly inhibit the proliferation of rat corneal stromal cell by down regulating TGF-β1 expression, therefore, it has potential prospect in lightening the corneal wound healing reaction.

  15. EFFECT OF HUMAN AMNIOTIC MEMBRANE ON CORNEAL EPITHELIUM AND YAC-1 CELL

    Institute of Scientific and Technical Information of China (English)

    叶纹; 沈玺; 钟一声

    2003-01-01

    Objective To study the effect of the amniotic membrane on enhancing the proliferation of corneal epithelia and YAC 1 cell.MethodsAfter the primary culture of the rabbits corneal epithelia and YAC 1 cells, they were seeded on the upper surface or stromal matrix side of amniotic membrane respectively. The proliferation results were observed by MTT test.ResultsThe amniotic membrane was found significantly enhancing the proliferation of corneal epithelia on the d1,d3,and d5 after culture. The proliferation rate was 28.93%,23.32%,23.41%(P<0.05)respectively, but the d7 proliferation rate was 20.72%(P>0.05).On the d1,d3,d7 after culture,the YAC 1 cells proliferation rate was 34.87%,36.28%,33.86%(P<0.01)respectively.ConclusionOur results demonstrated that the amniotic membrane could enhance the prolifera tion of both corneal epithelia and YAC 1 cells significantly. Although amniotic membrane has been suggested as an ideal material for reconstruction of ocular surface, special attention should be paid during amniotic membrane transplantation for treating ocular surface lesion resulted from epibulbar tumors.

  16. In Vitro Effects of Preserved and Unpreserved Anti-Allergic Drugs on Human Corneal Epithelial Cells

    OpenAIRE

    Guzman-Aranguez, Ana; Calvo, Patricia; Ropero, Inés; Pintor, Jesús

    2014-01-01

    Purpose: Treatment with topical eye drops for long-standing ocular diseases like allergy can induce detrimental side effects. The purpose of this study was to investigate in vitro cytotoxicity of commercially preserved and unpreserved anti-allergic eye drops on the viability and barrier function of monolayer and stratified human corneal-limbal epithelial cells.

  17. The effect of Lamium album extract on cultivated human corneal epithelial cells (10.014 pRSV-T

    Directory of Open Access Journals (Sweden)

    Roman Paduch

    2015-01-01

    Conclusion: Selected Lamium album extracts influence human corneal epithelial cells. Generally, while not toxic, they modulate pro-inflammatory and anti-inflammatory cytokines levels, and decrease NO release by cells; moreover, ethanol and ethyl acetate extracts reduce ROS levels.

  18. High-density lipoprotein endocytosis in endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Stefanie; Fruhwürth; Margit; Pavelka; Robert; Bittman; Werner; J; Kovacs; Katharina; M; Walter; Clemens; Rhrl; Herbert; Stangl

    2013-01-01

    AIM: To describe the way stations of high-density lipoprotein(HDL) uptake and its lipid exchange in endothelial cells in vitro and in vivo. METHODS: A combination of fluorescence microscopy using novel fluorescent cholesterol surrogates and electron microscopy was used to analyze HDL endocytosis in great detail in primary human endothelial cells. Further, HDL uptake was quantified using radio-labeled HDL particles. To validate the in vitro findings mice were injected with fluorescently labeled HDL and particle uptake in the liver was analyzed using fluorescencemicroscopy. RESULTS: HDL uptake occurred via clathrin-coated pits, tubular endosomes and multivesicular bodies in human umbilical vein endothelial cells. During uptake and resecretion, HDL-derived cholesterol was exchanged at a faster rate than cholesteryl oleate, resembling the HDL particle pathway seen in hepatic cells. In addition, lysosomes were not involved in this process and thus HDL degradation was not detectable. In vivo, we found HDL mainly localized in mouse hepatic endothelial cells. HDL was not detected in parenchymal liver cells, indicating that lipid transfer from HDL to hepatocytes occurs primarily via scavenger receptor, class B, type Ⅰ mediated selective uptake without concomitant HDL endocytosis. CONCLUSION: HDL endocytosis occurs via clathrincoated pits, tubular endosomes and multivesicular bodies in human endothelial cells. Mouse endothelial cells showed a similar HDL uptake pattern in vivo indicating that the endothelium is one major site of HDL endocytosis and transcytosis.

  19. Endothelial cell tumor growth is Ape/ref-1 dependent.

    Science.gov (United States)

    Biswas, Ayan; Khanna, Savita; Roy, Sashwati; Pan, Xueliang; Sen, Chandan K; Gordillo, Gayle M

    2015-09-01

    Tumor-forming endothelial cells have highly elevated levels of Nox-4 that release H2O2 into the nucleus, which is generally not compatible with cell survival. We sought to identify compensatory mechanisms that enable tumor-forming endothelial cells to survive and proliferate under these conditions. Ape-1/ref-1 (Apex-1) is a multifunctional protein that promotes DNA binding of redox-sensitive transcription factors, such as AP-1, and repairs oxidative DNA damage. A validated mouse endothelial cell (EOMA) tumor model was used to demonstrate that Nox-4-derived H2O2 causes DNA oxidation that induces Apex-1 expression. Apex-1 functions as a chaperone to keep transcription factors in a reduced state. In EOMA cells Apex-1 enables AP-1 binding to the monocyte chemoattractant protein-1 (mcp-1) promoter and expression of that protein is required for endothelial cell tumor formation. Intraperitoneal injection of the small molecule inhibitor E3330, which specifically targets Apex-1 redox-sensitive functions, resulted in a 50% decrease in tumor volume compared with mice injected with vehicle control (n = 6 per group), indicating that endothelial cell tumor proliferation is dependent on Apex-1 expression. These are the first reported results to establish Nox-4 induction of Apex-1 as a mechanism promoting endothelial cell tumor formation.

  20. Activation of Endothelial Nitric Oxide (eNOS Occurs through Different Membrane Domains in Endothelial Cells.

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    Jason Tran

    Full Text Available Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC with cholesterol and the oxysterol 7-ketocholesterol (7KC. Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1 colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells.

  1. Multidrug resistance-associated protein (MRP1, 2, 4 and 5) expression in human corneal cell culture models and animal corneal tissue.

    Science.gov (United States)

    Verstraelen, Jessica; Reichl, Stephan

    2014-07-07

    Preclinical studies addressing the transcorneal absorption of ophthalmic drugs are mainly performed using ex vivo animal corneas and in vitro corneal cell culture models, leaving open the question of transferability to humans in an in vivo situation. While passive drug absorption through corneal tissue is well understood, little is known about the expression of transporter proteins and active drug transport in human and animal corneas as well as corneal cell culture models. Therefore, the aim of this study was to conduct an expression analysis of four multidrug resistance-associated proteins (MRP1, 2, 4 and 5) in various in vitro and ex vivo corneal models, leading to a better understanding of the comparability of different corneal models regarding drug absorption and transferability to humans. Two well-established in vitro human corneal models, the HCE-T epithelial model and the more organotypic Hemicornea construct, both of which are based on the SV40 immortalized human corneal epithelial cell line HCE-T, were analyzed, as were excised rabbit and porcine cornea. Specimens of abraded epithelia from human donor corneas were also tested. MRP mRNA expression was determined via reverse transcriptase polymerase chain reaction. Protein expression was examined using Western blot experiments and immunohistochemistry. The functional activity of the MRP efflux transporter was detected in transport assays using specific marker and inhibitor substances. The functional expression of all of the tested MRP transporters was detected in the HCE-T epithelial model. Hemicornea constructs displayed a similar expression pattern for MRP1, 4 and 5, whereas no MRP2 protein expression or activity was detected. However, excised animal corneas exhibited different expression profiles. In porcine cornea, no functional expression of MRP1, 2, or 5 was observed, and we failed to detect MRP4 expression in rabbit cornea. The results suggest that MRP1, 2, 4, and 5 are expressed in the human corneal

  2. Differential expression of epithelial basement membrane components nidogens and perlecan in corneal stromal cells in vitro.

    Science.gov (United States)

    Santhanam, Abirami; Torricelli, Andre A M; Wu, Jiahui; Marino, Gustavo K; Wilson, Steven E

    2015-01-01

    The purpose of this study was to examine the expression of corneal epithelial basement membrane (EBM) components in different corneal stromal cell types. In vitro model systems were used to explore the expression of EBM components nidogen-1, nidogen-2, and perlecan that are the primary components in the lamina lucida and the lamina densa that defectively regenerate in corneas with stromal opacity after in -9.0 D photorefractive keratectomy (PRK). Primary rabbit corneal stromal cells were cultured using varying serum concentrations and exogenous growth factors, including fibroblast growth factor (FGF)-2 and transforming growth factor (TGF)-β1, to optimize the growth of each cell type of interest. The expression of the keratocyte-specific marker keratocan and the myofibroblast-specific marker α-smooth muscle actin (α-SMA) were analyzed with real-time PCR, western blot, and immunocytochemical staining to evaluate the specificity of the cell types and select optimal conditions (high keratocan and low α-SMA for keratocytes; low keratocan and high α-SMA for myofibroblasts; low keratocan and low α-SMA for corneal fibroblasts). The expression of the EBM components nidogen-1, nidogen-2, and perlecan was evaluated in each corneal cell type using real-time PCR, immunostaining, and western blotting. In agreement with previous studies, serum-free DMEM was found to be optimal for keratocytes, DMEM with 10% serum and 40 ng/ml FGF-2 yielded the best marker profile for corneal fibroblasts, and DMEM with 1% serum and 2 ng/ml TGF-β1 was found to be optimal for myofibroblasts. Nidogen-1 and nidogen-2 mRNAs were highly expressed in keratocytes, whereas perlecan was highly expressed in myofibroblasts. In keratocytes, nidogen-2 and perlecan proteins were expressed predominantly in intracellular compartments, whereas in myofibroblasts expression of both EBM components was observed diffusely throughout the cell. Although the perlecan mRNA levels were high in the myofibroblasts, the

  3. Growth of fibroblasts and endothelial cells on wettability gradient surfaces

    NARCIS (Netherlands)

    Ruardy, TG; Moorlag, HE; Schakenraad, JM; VanderMei, HC; Busscher, HJ

    1997-01-01

    The growth, spreading, and shape of human skin fibroblasts (PK 84) and human umbilical cord endothelial cells on dichlorodimethylsilane (DDS) and dimethyloctadecylchlorosilane (DOGS) gradient surfaces were investigated in the presence of serum proteins. Gradient surfaces were prepared on glass using

  4. Endothelial cell proliferation in swine experimental aneurysm after coil embolization.

    Directory of Open Access Journals (Sweden)

    Yumiko Mitome-Mishima

    Full Text Available After coil embolization, recanalization in cerebral aneurysms adversely influences long-term prognosis. Proliferation of endothelial cells on the coil surface may reduce the incidence of recanalization and further improve outcomes after coil embolization. We aimed to map the expression of proliferating tissue over the aneurysmal orifice and define the temporal profile of tissue growth in a swine experimental aneurysm model. We compared the outcomes after spontaneous thrombosis with those of coil embolization using histological and morphological techniques. In aneurysms that we not coiled, spontaneous thrombosis was observed, and weak, easily detachable proliferating tissue was evident in the aneurysmal neck. In contrast, in the coil embolization group, histological analysis showed endothelial-like cells lining the aneurysmal opening. Moreover, immunohistochemical and morphological analysis suggested that these cells were immature endothelial cells. Our results indicated the existence of endothelial cell proliferation 1 week after coil embolization and showed immature endothelial cells in septal tissue between the systemic circulation and the aneurysm. These findings suggest that endothelial cells are lead to and proliferate in the former aneurysmal orifice. This is the first examination to evaluate the temporal change of proliferating tissue in a swine experimental aneurysm model.

  5. Tumor-derived circulating endothelial cell clusters in colorectal cancer.

    KAUST Repository

    Cima, Igor

    2016-06-29

    Clusters of tumor cells are often observed in the blood of cancer patients. These structures have been described as malignant entities for more than 50 years, although their comprehensive characterization is lacking. Contrary to current consensus, we demonstrate that a discrete population of circulating cell clusters isolated from the blood of colorectal cancer patients are not cancerous but consist of tumor-derived endothelial cells. These clusters express both epithelial and mesenchymal markers, consistent with previous reports on circulating tumor cell (CTC) phenotyping. However, unlike CTCs, they do not mirror the genetic variations of matched tumors. Transcriptomic analysis of single clusters revealed that these structures exhibit an endothelial phenotype and can be traced back to the tumor endothelium. Further results show that tumor-derived endothelial clusters do not form by coagulation or by outgrowth of single circulating endothelial cells, supporting a direct release of clusters from the tumor vasculature. The isolation and enumeration of these benign clusters distinguished healthy volunteers from treatment-naïve as well as pathological early-stage (≤IIA) colorectal cancer patients with high accuracy, suggesting that tumor-derived circulating endothelial cell clusters could be used as a means of noninvasive screening for colorectal cancer. In contrast to CTCs, tumor-derived endothelial cell clusters may also provide important information about the underlying tumor vasculature at the time of diagnosis, during treatment, and throughout the course of the disease.

  6. Cellular and molecular biology of aging endothelial cells.

    Science.gov (United States)

    Donato, Anthony J; Morgan, R Garrett; Walker, Ashley E; Lesniewski, Lisa A

    2015-12-01

    Cardiovascular disease (CVD) is the leading cause of death in the United States and aging is a major risk factor for CVD development. One of the major age-related arterial phenotypes thought to be responsible for the development of CVD in older adults is endothelial dysfunction. Endothelial function is modulated by traditional CVD risk factors in young adults, but advancing age is independently associated with the development of vascular endothelial dysfunction. This endothelial dysfunction results from a reduction in nitric oxide bioavailability downstream of endothelial oxidative stress and inflammation that can be further modulated by traditional CVD risk factors in older adults. Greater endothelial oxidative stress with aging is a result of augmented production from the intracellular enzymes NADPH oxidase and uncoupled eNOS, as well as from mitochondrial respiration in the absence of appropriate increases in antioxidant defenses as regulated by relevant transcription factors, such as FOXO. Interestingly, it appears that NFkB, a critical inflammatory transcription factor, is sensitive to this age-related endothelial redox change and its activation induces transcription of pro-inflammatory cytokines that can further suppress endothelial function, thus creating a vicious feed-forward cycle. This review will discuss the two macro-mechanistic processes, oxidative stress and inflammation, that contribute to endothelial dysfunction with advancing age as well as the cellular and molecular events that lead to the vicious cycle of inflammation and oxidative stress in the aged endothelium. Other potential mediators of this pro-inflammatory endothelial phenotype are increases in immune or senescent cells in the vasculature. Of note, genomic instability, telomere dysfunction or DNA damage has been shown to trigger cell senescence via the p53/p21 pathway and result in increased inflammatory signaling in arteries from older adults. This review will discuss the current state

  7. Breast cancer cells stimulate osteoprotegerin (OPG production by endothelial cells through direct cell contact

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    Holen Ingunn

    2009-07-01

    Full Text Available Abstract Background Angiogenesis, the sprouting of capillaries from existing blood vessels, is central to tumour growth and progression, however the molecular regulation of this process remains to be fully elucidated. The secreted glycoprotein osteoprotegerin (OPG is one potential pro-angiogenic factor, and clinical studies have demonstrated endothelial cells within a number of tumour types to express high levels of OPG compared to those in normal tissue. Additionally, OPG can increase endothelial cell survival, proliferation and migration, as well as induce endothelial cell tube formation in vitro. This study aims to elucidate the processes involved in the pro-angiogenic effects of OPG in vitro, and also how OPG levels may be regulated within the tumour microenvironment. Results It has previously been demonstrated that OPG can induce tube formation on growth factor reduced matrigel. In this study, we demonstrate that OPG enhances the pro-angiogenic effects of VEGF and that OPG does not stimulate endothelial cell tube formation through activation of the VEGFR2 receptor. We also show that cell contact between HuDMECs and the T47D breast cancer cell line increases endothelial cell OPG mRNA and protein secretion levels in in vitro co-cultures. These increases in endothelial cell OPG secretion were dependent on ανβ3 ligation and NFκB activation. In contrast, the pro-angiogenic factors VEGF, bFGF and TGFβ had no effect on HuDMEC OPG levels. Conclusion These findings suggest that the VEGF signalling pathway is not involved in mediating the pro-angiogenic effects of OPG on endothelial cells in vitro. Additionally, we show that breast cancer cells cause increased levels of OPG expression by endothelial cells, and that direct contact between endothelial cells and tumour cells is required in order to increase endothelial OPG expression and secretion. Stimulation of OPG secretion was shown to involve ανβ3 ligation and NFκB activation.

  8. Uptake of gold nanoparticles in primary human endothelial cells

    DEFF Research Database (Denmark)

    Klingberg, Henrik; Oddershede, Lene B.; Löschner, Katrin

    2015-01-01

    Gold nanoparticles (AuNPs) are relevant in nanomedicine for drug delivery in the vascular system, where endothelial cells are the first point of contact. We investigated the uptake of 80 nm AuNPs in primary human umbilical vein endothelial cells (HUVECs) by flow cytometry, 3D confocal microscopy....... Uptake of AuNPs in HUVECs occurred mainly by clathrin-mediated endocytosis and trafficking to membrane enclosures in the form of single particles and agglomerates of 2–3 particles....

  9. Endothelial cell apoptosis correlates with low haptoglobin concentrations in diabetes.

    Science.gov (United States)

    Dalan, Rinkoo; Liu, Xiaofeng; Goh, Liuh Ling; Bing, Sun; Luo, Kathy Qian

    2017-08-01

    The haptoglobin 2-2 genotype is associated with lower haptoglobin concentrations and atherosclerosis in diabetes. Endothelial cell apoptosis contributes significantly to atherosclerosis. We studied endothelial cell apoptosis in diabetes patients with haptoglobin 2-2 and non-haptoglobin 2-2 genotype. Approach and results: We pooled plasma from 10 patients with haptoglobin 2-2 and non-haptoglobin 2-2 genotype and quantified endothelial cell apoptosis using a hemodynamic lab-on-chip system. Then, we conducted similar experiments on individual diabetes plasma samples with the haptoglobin 2-2 ( n = 20) and non-haptoglobin 2-2 genotype ( n = 20). Haptoglobin beta concentrations were measured by Western blot analysis. We looked for association with demographic, metabolic variables, inflammation and oxidative stress. In pooled plasma, endothelial cell apoptosis was higher in haptoglobin 2-2 group (haptoglobin 2-2: 23.18% vs non-haptoglobin 2-2:15.32%). In individual samples, univariate analysis showed that endothelial cell apoptosis correlated with haptoglobin beta concentration [ β = -10.29 (95% confidence interval: -13.44, -7.14), p  0.05). These results show that regardless of the haptoglobin genotype, haptoglobin is associated with prevention of endothelial cell apoptosis in diabetes.

  10. Traction Forces of Endothelial Cells under Slow Shear Flow

    Science.gov (United States)

    Perrault, Cecile M.; Brugues, Agusti; Bazellieres, Elsa; Ricco, Pierre; Lacroix, Damien; Trepat, Xavier

    2015-01-01

    Endothelial cells are constantly exposed to fluid shear stresses that regulate vascular morphogenesis, homeostasis, and disease. The mechanical responses of endothelial cells to relatively high shear flow such as that characteristic of arterial circulation has been extensively studied. Much less is known about the responses of endothelial cells to slow shear flow such as that characteristic of venous circulation, early angiogenesis, atherosclerosis, intracranial aneurysm, or interstitial flow. Here we used a novel, to our knowledge, microfluidic technique to measure traction forces exerted by confluent vascular endothelial cell monolayers under slow shear flow. We found that cells respond to flow with rapid and pronounced increases in traction forces and cell-cell stresses. These responses are reversible in time and do not involve reorientation of the cell body. Traction maps reveal that local cell responses to slow shear flow are highly heterogeneous in magnitude and sign. Our findings unveil a low-flow regime in which endothelial cell mechanics is acutely responsive to shear stress. PMID:26488643

  11. Glucose transporter 1-positive endothelial cells in infantile hemangioma exhibit features of facultative stem cells

    Science.gov (United States)

    Huang, Lan; Nakayama, Hironao; Klagsbrun, Michael; Mulliken, John B.; Bischoff, Joyce

    2014-01-01

    Endothelial glucose transporter 1 (GLUT1) is a definitive and diagnostic marker for infantile hemangioma (IH), a vascular tumor of infancy. To date, GLUT1-positive endothelial cells in IH have not been quantified nor directly isolated and studied. We isolated GLUT1-positive and GLUT1-negative endothelial cells from IH specimens and characterized their proliferation, differentiation and response to propranolol, a first-line therapy for IH, and to rapamycin, an mTOR pathway inhibitor used to treat an increasingly wide array of proliferative disorders. Although freshly isolated GLUT1-positive cells, selected using anti-GLUT1 magnetic beads, expressed endothelial markers CD31, VE-Cadherin and VEGFR2, they converted to a mesenchymal phenotype after three weeks in culture. In contrast, GLUT1-negative endothelial cells exhibited a stable endothelial phenotype in vitro. GLUT1-selected cells were clonogenic when plated as single cells and could be induced to re-differentiate into endothelial cells, or into pericyte/smooth muscle cells or into adipocytes, indicating a stem cell-like phenotype. These data demonstrate that, although they appear and function in the tumor as bona fide endothelial cells, the GLUT1-positive endothelial cells display properties of facultative stem cells. Pretreatment with rapamycin for 4 days significantly slowed proliferation of GLUT1-selected cells, whereas propranolol pretreatment had no effect. These results reveal for the first time the facultative nature of GLUT1-positive endothelial cells in infantile hemangioma. PMID:25187207

  12. Endothelial keratoplasty: evolution and horizons

    Directory of Open Access Journals (Sweden)

    Gustavo Teixeira Grottone

    2012-12-01

    Full Text Available Endothelial keratoplasty has been adopted by corneal surgeons worldwide as an alternative to penetrating keratoplasty (PK in the treatment of corneal endothelial disorders. Since the first surgeries in 1998, different surgical techniques have been used to replace the diseased endothelium. Compared with penetrating keratoplasty, all these techniques may provide faster and better visual rehabilitation with minimal change in refractive power of the transplanted cornea, minimal induced astigmatism, elimination of suture-induced complications and late wound dehiscence, and a reduced demand for postoperative care. Translational research involving cell-based therapy is the next step in work on endothelial keratoplasty. The present review updates information on comparisons among different techniques and predicts the direction of future treatment.

  13. Endothelial induced EMT in breast epithelial cells with stem cell properties

    DEFF Research Database (Denmark)

    Sigurdsson, Valgardur; Hilmarsdottir, Bylgja; Sigmundsdottir, Hekla

    2011-01-01

    endothelial cells might play a role in EMT. Using a 3D culture model we demonstrate that endothelial cells are potent inducers of EMT in D492 an immortalized breast epithelial cell line with stem cell properties. Endothelial induced mesenchymal-like cells (D492M) derived from D492, show reduced expression...... to the vascular rich areas show no or decreased expression of E-Cad and increased N-Cad expression suggesting EMT. Collectively, we have shown in a 3D culture model that endothelial cells are potent inducers of EMT in breast epithelial cells with stem cell properties. Furthermore, we demonstrate that basal...

  14. Evaluation of corneal cell growth on tissue engineering materials as artificial cornea scaffolds

    Directory of Open Access Journals (Sweden)

    Hai-Yan Wang

    2013-12-01

    Full Text Available The keratoprosthesis (KPro; artificial cornea is a special refractive device to replace human cornea by using heterogeneous forming materials for the implantation into the damaged eyes in order to obtain a certain vision. The main problems of artificial cornea are the biocompatibility and stability of the tissue particularly in penetrating keratoplasty. The current studies of tissue-engineered scaffold materials through comprising composites of natural and synthetic biopolymers together have developed a new way to artificial cornea. Although a wide agreement that the long-term stability of these devices would be greatly improved by the presence of cornea cells, modification of keratoprosthesis to support cornea cells remains elusive. Most of the studies on corneal substrate materials and surface modification of composites have tried to improve the growth and biocompatibility of cornea cells which can not only reduce the stimulus of heterogeneous materials, but also more importantly continuous and stable cornea cells can prevent the destruction of collagenase. The necrosis of stroma and spontaneous extrusion of the device, allow for maintenance of a precorneal tear layer, and play the role of ensuring a good optical surface and resisting bacterial infection. As a result, improvement in corneal cells has been the main aim of several recent investigations; some effort has focused on biomaterial for its well biological properties such as promoting the growth of cornea cells. The purpose of this review is to summary the growth status of the corneal cells after the implantation of several artificial corneas.

  15. TSG-6 protects corneal endothelium from transcorneal cryoinjury in rabbits.

    Science.gov (United States)

    Kim, Jeong-Ah; Ko, Jung Hwa; Ko, Ah Young; Lee, Hyun Ju; Kim, Mee Kum; Wee, Won Ryang; Lee, Ryang Hwa; Fulcher, Samuel F; Oh, Joo Youn

    2014-07-17

    To investigate the effect of an anti-inflammatory protein, TNF-α stimulated gene/protein (TSG)-6 and an antiapoptotic protein, stanniocalcin (STC)-1 on corneal endothelium in rabbits with transcorneal cryoinjury. Transcorneal freezing (-80°C) was applied to rabbit corneas for 30 seconds. Immediately post injury, either TSG-6 (10 μg/100 μL), STC-1 (10 μg/100 μL), or the same volume of balanced salt solution (BSS) was injected into the anterior chamber. Each eye was examined for corneal opacity, corneal thickness, endothelial cell density, and endothelial hexagonality every 2 to 6 hours for 48 hours post injury. The concentrations of myeloperoxidase (MPO) and IL-1β were measured in the aqueous humor every 6 hours. At 48 hours post injury, each cornea was assayed for TNF-α, IL-1β, IL-6, and MPO, and histologically evaluated with alizarin red-trypan blue staining, hematoxylin-eosin staining, and immunostaining for neutrophils. Tumor necrosis factor-α stimulated gene/protein-6 significantly decreased the development of corneal opacity and edema after cryoinjury compared with STC-1 or BSS. The corneal endothelial cell density and hexagonality were markedly preserved by TSG-6. The mRNA levels of TNF-α, IL-1β, and IL-6 in the cornea and the protein levels of MPO and IL-1β in the aqueous humor and cornea were significantly lower in TSG-6-treated eyes than BSS-treated controls. Similarly, the expression of fibroblast growth factor-2 was reduced by TSG-6 treatment. Histologic evaluation demonstrated that neutrophil infiltration of the cornea was decreased in TSG-6-treated eyes. Tumor necrosis factor-α stimulated gene/protein-6 protected corneal endothelial cells from transcorneal cryoinjury through suppression of inflammation. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

  16. Corneal decompensation following filtering surgery with the Ex-PRESS® mini glaucoma shunt device

    Directory of Open Access Journals (Sweden)

    Tojo N

    2015-03-01

    Full Text Available Naoki Tojo, Atsushi Hayashi, Akio Miyakoshi Department of Ophthalmology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan Purpose: To report a case of corneal decompensation due to the Ex-PRESS® mini glaucoma shunt device (Ex-PRESS.Patient and methods: A 75-year-old man had pseudoexfoliation glaucoma in his right eye. He underwent filtration surgery with Ex-PRESS. His intraocular pressure was 7 mmHg after 9 months.Results: We observed partial decompensation of the corneal endothelium adjacent to the filtering bleb. Specular microscopy revealed a marked decrease in the endothelial cell density at the center of the cornea.Conclusion: Anterior segment optical coherence tomography is very useful for evaluating corneal edema and the position of Ex-PRESS. It is important to follow up with an examination of the corneal endothelial cells. Keywords: Ex-PRESS, bullous keratopathy, trabeculectomy, complication, cornea 

  17. Erythropoietin improves cardiac function through endothelial progenitor cell and vascular endothelial growth factor mediated neovascularization

    NARCIS (Netherlands)

    Westenbrink, B. Daan; Lipsic, Erik; van der Meer, Peter; van der Harst, Pirn; Oeseburg, Hisko; Sarvaas, Gideon J. Du Marchie; Koster, Johan; Voors, Adriaan A.; van Veldhuisen, Dirk J.; van Gilst, Wiek H.; Schoemaker, Regien G.

    2007-01-01

    Aims Erythropoietin (EPO) improves cardiac function and induces neovascutarization in chronic heart failure (CHF), although the exact mechanism has not been elucidated. We studied the effects of EPO on homing and incorporation of endothelial progenitor cells (EPC) into the myocardial microvasculatur

  18. Triazole RGD antagonist reverts TGFβ1-induced endothelial-to-mesenchymal transition in endothelial precursor cells.

    Science.gov (United States)

    Bianchini, Francesca; Peppicelli, Silvia; Fabbrizzi, Pierangelo; Biagioni, Alessio; Mazzanti, Benedetta; Menchi, Gloria; Calorini, Lido; Pupi, Alberto; Trabocchi, Andrea

    2017-01-01

    Fibrosis is the dramatic consequence of a dysregulated reparative process in which activated fibroblasts (myofibroblasts) and Transforming Growth Factor β1 (TGFβ1) play a central role. When exposed to TGFβ1, fibroblast and epithelial cells differentiate in myofibroblasts; in addition, endothelial cells may undergo endothelial-to-mesenchymal transition (EndoMT) and actively participate to the progression of fibrosis. Recently, the role of αv integrins, which recognize the Arg-Gly-Asp (RGD) tripeptide, in the release and signal transduction activation of TGFβ1 became evident. In this study, we present a class of triazole-derived RGD antagonists that interact with αvβ3 integrin. Above different compounds, the RGD-2 specifically interferes with integrin-dependent TGFβ1 EndoMT in Endothelial Colony-Forming Cells (ECPCs) derived from circulating Endothelial Precursor Cells (ECPCs). The RGD-2 decreases the amount of membrane-associated TGFβ1, and reduces both ALK5/TGFβ1 type I receptor expression and Smad2 phosphorylation in ECPCs. We found that RGD-2 antagonist reverts EndoMT, reducing α-smooth muscle actin (α-SMA) and vimentin expression in differentiated ECPCs. Our results outline the critical role of integrin in fibrosis progression and account for the opportunity of using integrins as target for anti-fibrotic therapeutic treatment.

  19. Roles of TRIM32 in Corneal Epithelial Cells After Infection with Herpes Simplex Virus

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    Hao Cui

    2017-09-01

    Full Text Available Background: Epithelial cells play important roles as a critical barrier in protecting the cornea from microbial pathogens infection. Methods: In this study, we were aiming to investigate the role of E3 ubiquitin ligase tripartite motif protein 32 (TRIM32 in corneal epithelial cells in response to Herpes Simplex Virus type 1 (HSV-1 infection and to elucidate the underlying mechanisms. Results: We found the expression of TRIM32 was increased after infected with HSV-1 both in murine corneas and cultured human epithelial (HCE cells. Furthermore, knockdown of the expression of TRIM32 significantly aggravated HSV-1 induced herpetic stromal keratitis (HSK in mice and promoted the replication of HSV-1 in cultured HCE cells. We also observed that silencing of TRIM32 resulted in the decreased expression of IFN-β and suppressed activation of interferon regulatory factor 3 (IRF3 both in vivo and in vitro. Finally, we found TRIM32 positively regulate IFN-β production in corneal epithelial cells through promoting K63-linked polyubiquitination of stimulator of interferon genes (STING. Conclusion: In conclusion, our data suggested that TRIM32 as a crucial positive regulator of HSV-1 induced IFN-β production in corneal epithelial cells, and it played a predominant role in clearing HSV-1 from the cornea.

  20. Case of late-onset corneal decompensation after iris-fixated phakic intraocular lens implantation.

    NARCIS (Netherlands)

    Eijden, R. van; Vries, N.E. de; Cruysberg, L.P.J.; Webers, C.A.; Berenschot, T.; Nuijts, R.M.

    2009-01-01

    A 48-year-old myopic patient with bilateral anterior chamber depth of 3.1 mm and endothelial cell density (ECD) of 2525 cells/mm(2) and 2638 cells/mm(2) preoperatively had bilateral implantation of an Artisan iris-fixated phakic intraocular lens (pIOL). Five years postoperatively, unilateral corneal

  1. Human iPSC-Derived Endothelial Cell Sprouting Assay in ...

    Science.gov (United States)

    Activation of vascular endothelial cells (ECs) by growth factors initiates a cascade of events in vivo consisting of EC tip cell selection, sprout formation, EC stalk cell proliferation, and ultimately vascular stabilization by support cells. Although EC functional assays can recapitulate one or more aspects of angiogenesis in vitro, they are often limited by a lack of definition to the substratum and lack of dependence on key angiogenic signaling axes. Here, we designed and characterized a chemically-defined model of endothelial sprouting behavior in vitro using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs). Thiol-ene photopolymerization was used to rapidly encapsulate iPSC-ECs at high density in poly(ethylene glycol) (PEG) hydrogel spheres and subsequently to rapidly encapsulate iPSC-EC-containing hydrogel spheres in a cell-free over-layer. The hydrogel sprouting array here maintained pro-angiogenic phenotype of iPSC-ECs and supported growth factor-dependent proliferation and sprouting behavior. The sprouting model responded appropriately to several reference pharmacological angiogenesis inhibitors, which suggests the functional role of vascular endothelial growth factor, NF-κB, matrix metalloproteinase-2/9, protein kinase activity, and β-tubulin in endothelial sprouting. A blinded screen of 38 putative vascular disrupting compounds (pVDCs) from the US Environmental Protection Agency’s ToxCast library identified five compounds th

  2. Endothelial Progenitor Cells for Diagnosis and Prognosis in Cardiovascular Disease

    OpenAIRE

    2015-01-01

    Objective. To identify, evaluate, and synthesize evidence on the predictive power of circulating endothelial progenitor cells (EPCs) in cardiovascular disease, through a systematic review of quantitative studies. Data Sources. MEDLINE was searched using keywords related to “endothelial progenitor cells” and “endothelium” and, for the different categories, respectively, “smoking”; “blood pressure”; “diabetes mellitus” or “insulin resistance”; “dyslipidemia”; “aging” or “elderly”; “angina p...

  3. Synergistic effects of telmisartan and simvastatin on endothelial progenitor cells

    OpenAIRE

    Steinmetz, Martin; Brouwers, Caroline; Nickenig, Georg; Wassmann, Sven

    2009-01-01

    Abstract Circulating endothelial progenitor cells (EPC) contribute to endothelial replenishment. Telmisartan is an angiotensin-receptor blocker with PPARγ-agonistic properties. PPARγ-agonists and HMG-CoA reductase inhibitors have been shown to enhance EPC number and function. We focused on the effects of telmisartan alone or in combination with simvastatin on EPC. EPC were isolated from healthy human volunteers, cultured and stimulated with telmisartan, simvastatin, or the combination of telm...

  4. Metformin improves endothelial function in aortic tissue and microvascular endothelial cells subjected to diabetic hyperglycaemic conditions.

    Science.gov (United States)

    Ghosh, Suparna; Lakshmanan, Arun P; Hwang, Mu Ji; Kubba, Haidar; Mushannen, Ahmed; Triggle, Chris R; Ding, Hong

    2015-12-01

    The cellular mechanisms whereby metformin, the first line drug for type 2 diabetes (T2DM), mediates its antidiabetic effects remain elusive, particularly as to whether metformin has a direct protective action on the vasculature. This study was designed to determine if a brief 3-h exposure to metformin protects endothelial function against the effects of hyperglycaemia. We investigated the protective effects of metformin on endothelial-dependent vasodilatation (EDV) in thoracic aortae from T2DM db/db mice and on high glucose (HG, 40 mM) induced changes in endothelial nitric oxide synthase (eNOS) signaling in mouse microvascular endothelial cells (MMECs) in culture. Exposure of aortae from db+/? non-diabetic control mice to high glucose (HG, 40 mM) containing Krebs for 3-h significantly (Pmetformin; metformin also improved ACh-induced EDV in aortae from diabetic db/db mice. Immunoblot analysis of MMECs cultured in HG versus NG revealed a significant reduction of the ratio of phosphorylated (p-eNOS)/eNOS and p-Akt/Akt, but not the expression of total eNOS or Akt. The 3-h exposure of MMECs to metformin significantly (Pmetformin can reverse/reduce the impact of HG on endothelial function, via mechanisms linked to increased phosphorylation of eNOS and Akt.

  5. Cells from the adult corneal stroma can be reprogrammed to a neuron-like cell using exogenous growth factors

    Energy Technology Data Exchange (ETDEWEB)

    Greene, Carol Ann, E-mail: carol.greene@auckland.ac.nz; Chang, Chuan-Yuan; Fraser, Cameron J.; Nelidova, Dasha E.; Chen, Jing A.; Lim, Angela; Brebner, Alex; McGhee, Jennifer; Sherwin, Trevor; Green, Colin R.

    2014-03-10

    Cells thought to be stem cells isolated from the cornea of the eye have been shown to exhibit neurogenic potential. We set out to uncover the identity and location of these cells within the cornea and to elucidate their neuronal protein and gene expression profile during the process of switching to a neuron-like cell. Here we report that every cell of the adult human and rat corneal stroma is capable of differentiating into a neuron-like cell when treated with neurogenic differentiation specifying growth factors. Furthermore, the expression of genes regulating neurogenesis and mature neuronal structure and function was increased. The switch from a corneal stromal cell to a neuron-like cell was also shown to occur in vivo in intact corneas of living rats. Our results clearly indicate that lineage specifying growth factors can affect changes in the protein and gene expression profiles of adult cells, suggesting that possibly many adult cell populations can be made to switch into another type of mature cell by simply modifying the growth factor environment. - Highlights: • Adult corneal stromal cells can differentiated into neuron-like cells. • Neuronal specification of the adult stromal cell population is stochastic. • Neuronal specification in an adult cell population can be brought about by growth factors.

  6. Multi-scale undulations in human aortic endothelial cell fibers.

    Science.gov (United States)

    Frketic, Jolie B; DeLaPeña, Abigail; Suaris, Melanie G; Zehnder, Steven M; Angelini, Thomas E

    2015-02-01

    Blood vessels often have an undulatory morphology, with excessive bending, kinking, and coiling occuring in diseased vasculature. The underlying physical causes of these morphologies are generally attributed, in combination, to changes in blood pressure, blood flow rate, and cell proliferation or apoptosis. However, pathological vascular morphologies often start during developmental vasculogenesis. At early stages of vasculogenesis, angioblasts (vascular endothelial cells that have not formed a lumen) assemble into primitive vessel-like fibers before blood flow occurs. If loose, fibrous aggregates of endothelial cells can generate multi-cellular undulations through mechanical instabilities, driven by the cytoskeleton, new insight into vasculature morphology may be achieved with simple in vitro models of endothelial cell fibers. Here we study mechanical instabilities in vessel-like structures made from endothelial cells embedded in a collagen matrix. We find that endothelial cell fibers contract radially over time, and undulate at two dominant wavelengths: approximately 1cm and 1mm. Simple mechanical models suggest that the long-wavelength undulation is Euler buckling in rigid confinement, while the short-wavelength buckle may arise from a mismatch between fiber bending energy and matrix deformation. These results suggest a combination of fiber-like geometry, cystoskeletal contractions, and extracellular matrix elasticity may contribute to undulatory blood vessel morphology in the absence of a lumen or blood pressure.

  7. Expression of Phospholipases A2 and C in Human Corneal Epithelial Cells

    Science.gov (United States)

    Landreville, Solange; Coulombe, Stéphanie; Carrier, Patrick; Gelb, Michael H.; Guérin, Sylvain L.; Salesse, Christian

    2008-01-01

    Purpose To achieve a better understanding of the involvement of phospholipases in the inflammation and wound-healing processes in human corneal epithelial cells (HCECs), expression of phospholipase A2s (PLA2s) and phospholipase Cs (PLCs) was examined in the human corneal epithelium. Methods Specific primers were designed for RT-PCR amplification of the known secreted (s)PLA2, cytosolic (c)PLA2, and PLC mRNAs. Corresponding PCR products were cloned and the DNA sequenced. Immunofluorescence of flatmounted corneal sections and Western blot analyses were used to detect the PLA2s and PLCs expressed by HCECs. Results The mRNAs for the following phospholipases were detected by RT-PCR in the HCECs: sPLA2GIII, -GX, and -GXIIA; cPLA2α and -γ; PLCβ1, -β2, -β3, -β4, -γ1, -γ2, -δ1, -δ3, -δ4, and -ε. Immunofluorescence analyses conducted on corneal epithelium cryosections and Western blot on freshly isolated HCECs demonstrated the presence of sPLA2GIII, -GX, and -GXIIA; cPLA2α and -γ; and PLCβ2, -β3, -γ1, -γ2, and -δ3. Conclusions Many phospholipase isoforms are expressed by HCECs and may play a major role in signal transduction (PLCs) as well as in the release of precursors of potent mediators of inflammation, such as leukotrienes and prostaglandins (PLA2s). Moreover, the sPLA2s expressed by the corneal epithelium could be involved in the normal antibacterial activity in the tears and in wound healing. PMID:15505048

  8. Induction of corneal epithelial progenitors from bone-marrow mesenchymal stem cells of rhesus monkeys in vitro

    Institute of Scientific and Technical Information of China (English)

    YUAN Jing; YU JianXiong; HUANG Bing; LIU BingQian; LIU JingBo; JIANG RuZhang; GE Jian

    2007-01-01

    Bioengineered corneas are substitutes for human donor tissue that are designed to treat severe disease affecting ocular surfaces.However, a shortage of candidate seed cells for bioengineering corneas is still a problem.Bone-marrow mesenchymal stem cells (MSCs) are capable of multilineage differentiation.Therefore, we determined whether MSCs differentiate into corneal epithelial cells (ECs).We applied three exoteric-microenvironmental systems to induce MSCs to become ECs.Induced MSC were identified by means of morphologic examination, immunocytochemical analysis, and flow cytometry.MSCs grown in one microenvironment had characteristics similar to those of corneal epithelial progenitors.Induced MSCs expressed markers for EC, including integrin β1, Cx43, Pax6, and P63.MSCs were successfully induced to become corneal epithelial progenitors.Therefore, the use of MSCs may hold substantial promise for reconstructing the ocular surface after corneal injury.

  9. Characterization of vascular endothelial progenitor cells from chicken bone marrow

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    Bai Chunyu

    2012-05-01

    Full Text Available Abstract Background Endothelial progenitor cells (EPC are a type of stem cell used in the treatment of atherosclerosis, vascular injury and regeneration. At present, most of the EPCs studied are from human and mouse, whereas the study of poultry-derived EPCs has rarely been reported. In the present study, chicken bone marrow-derived EPCs were isolated and studied at the cellular level using immunofluorescence and RT-PCR. Results We found that the majority of chicken EPCs were spindle shaped. The growth-curves of chicken EPCs at passages (P 1, -5 and -9 were typically “S”-shaped. The viability of chicken EPCs, before and after cryopreservation was 92.2% and 81.1%, respectively. Thus, cryopreservation had no obvious effects on the viability of chicken EPCs. Dil-ac-LDL and FITC-UAE-1 uptake assays and immunofluorescent detection of the cell surface markers CD34, CD133, VEGFR-2 confirmed that the cells obtained in vitro were EPCs. Observation of endothelial-specific Weibel-Palade bodies using transmission electron microscopy further confirmed that the cells were of endothelial lineage. In addition, chicken EPCs differentiated into endothelial cells and smooth muscle cells upon induction with VEGF and PDGF-BB, respectively, suggesting that the chicken EPCs retained multipotency in vitro. Conclusions These results suggest that chicken EPCs not only have strong self-renewal capacity, but also the potential to differentiate into endothelial and smooth muscle cells. This research provides theoretical basis and experimental evidence for potential therapeutic application of endothelial progenitor cells in the treatment of atherosclerosis, vascular injury and diabetic complications.

  10. Hypoxia, leptin, and vascular endothelial growth factor stimulate vascular endothelial cell differentiation of human adipose tissue-derived stem cells.

    Science.gov (United States)

    Bekhite, Mohamed M; Finkensieper, Andreas; Rebhan, Jennifer; Huse, Stephanie; Schultze-Mosgau, Stefan; Figulla, Hans-Reiner; Sauer, Heinrich; Wartenberg, Maria

    2014-02-15

    The plasticity of human adipose tissue-derived stem cells (hASCs) is promising, but differentiation in vitro toward endothelial cells is poorly understood. Flow cytometry demonstrated that hASCs isolated from excised fat tissue were positive for CD29, CD44, CD70, CD90, CD105, and CD166 and negative for the endothelial marker CD31, and the hematopoietic cell markers CD34 and CD133. hASCs differentiated into adipocytes after cultivation in adipogenic medium. Exposure of hASCs for 10 days under hypoxia (3% oxygen) in combination with leptin increased the percentage of CD31(+) endothelial cells as well as CD31, VE-Cadherin, Flk-1, Tie2, von Willebrand factor, and endothelial cell nitric oxide synthase mRNA expression. This was enhanced on co-incubation of vascular endothelial growth factor (VEGF) and leptin, whereas VEGF alone was not sufficient. Moreover, hASCs cultured on a matrigel surface under hypoxia/VEGF/leptin, showed a stable branching network. Hypoxic conditions significantly decreased apoptosis as evaluated by cleaved caspase-3, and increased prolyl hydroxylase domain 3 mRNA expression. Hypoxia increased expression of VEGF as well as leptin transcripts, which were significantly inhibited on co-incubation with either VEGF or leptin or a combination of both. Furthermore, leptin treatment of hypoxic cells increased the expression of the long/signaling form of the leptin receptor (ObRL), which was augmented on co-incubation with VEGF. The observed endothelial differentiation was dependent on the Akt pathway, as co-administration with Akt inhibitor abolished the observed effects. In conclusion, our data demonstrate that hASCs can be efficiently differentiated to endothelial cells by mimicking the hypoxic and pro-angiogenic microenvironment of adipose tissue.

  11. Endothelial cells regulate neural crest and second heart field morphogenesis.

    Science.gov (United States)

    Milgrom-Hoffman, Michal; Michailovici, Inbal; Ferrara, Napoleone; Zelzer, Elazar; Tzahor, Eldad

    2014-07-04

    Cardiac and craniofacial developmental programs are intricately linked during early embryogenesis, which is also reflected by a high frequency of birth defects affecting both regions. The molecular nature of the crosstalk between mesoderm and neural crest progenitors and the involvement of endothelial cells within the cardio-craniofacial field are largely unclear. Here we show in the mouse that genetic ablation of vascular endothelial growth factor receptor 2 (Flk1) in the mesoderm results in early embryonic lethality, severe deformation of the cardio-craniofacial field, lack of endothelial cells and a poorly formed vascular system. We provide evidence that endothelial cells are required for migration and survival of cranial neural crest cells and consequently for the deployment of second heart field progenitors into the cardiac outflow tract. Insights into the molecular mechanisms reveal marked reduction in Transforming growth factor beta 1 (Tgfb1) along with changes in the extracellular matrix (ECM) composition. Our collective findings in both mouse and avian models suggest that endothelial cells coordinate cardio-craniofacial morphogenesis, in part via a conserved signaling circuit regulating ECM remodeling by Tgfb1.

  12. Endothelial cells regulate neural crest and second heart field morphogenesis

    Directory of Open Access Journals (Sweden)

    Michal Milgrom-Hoffman

    2014-07-01

    Full Text Available Cardiac and craniofacial developmental programs are intricately linked during early embryogenesis, which is also reflected by a high frequency of birth defects affecting both regions. The molecular nature of the crosstalk between mesoderm and neural crest progenitors and the involvement of endothelial cells within the cardio–craniofacial field are largely unclear. Here we show in the mouse that genetic ablation of vascular endothelial growth factor receptor 2 (Flk1 in the mesoderm results in early embryonic lethality, severe deformation of the cardio–craniofacial field, lack of endothelial cells and a poorly formed vascular system. We provide evidence that endothelial cells are required for migration and survival of cranial neural crest cells and consequently for the deployment of second heart field progenitors into the cardiac outflow tract. Insights into the molecular mechanisms reveal marked reduction in Transforming growth factor beta 1 (Tgfb1 along with changes in the extracellular matrix (ECM composition. Our collective findings in both mouse and avian models suggest that endothelial cells coordinate cardio–craniofacial morphogenesis, in part via a conserved signaling circuit regulating ECM remodeling by Tgfb1.

  13. Fibroblast nemosis induces angiogenic responses of endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Enzerink, Anna, E-mail: anna.enzerink@helsinki.fi [Haartman Institute, University of Helsinki, P.O. BOX 21, FIN-00014 Helsinki (Finland); Rantanen, Ville, E-mail: ville.rantanen@helsinki.fi [Computational Systems Biology Laboratory, Institute of Biomedicine and Genome-Scale Biology Research Program, University of Helsinki, P.O. BOX 63, 00014 Helsinki (Finland); Vaheri, Antti, E-mail: antti.vaheri@helsinki.fi [Haartman Institute, University of Helsinki, P.O. BOX 21, FIN-00014 Helsinki (Finland)

    2010-03-10

    Increasing evidence points to a central link between inflammation and activation of the stroma, especially of fibroblasts therein. However, the mechanisms leading to such activation mostly remain undescribed. We have previously characterized a novel type of fibroblast activation (nemosis) where clustered fibroblasts upregulated the production of cyclooxygenase-2, secretion of prostaglandins, proteinases, chemotactic cytokines, and hepatocyte growth factor (HGF), and displayed activated nuclear factor-{kappa}B. Now we show that nemosis drives angiogenic responses of endothelial cells. In addition to HGF, nemotic fibroblasts secreted vascular endothelial growth factor (VEGF), and conditioned medium from spheroids promoted sprouting and networking of human umbilical venous endothelial cells (HUVEC). The response was partly inhibited by function-blocking antibodies against HGF and VEGF. Conditioned nemotic fibroblast medium promoted closure of HUVEC and human dermal microvascular endothelial cell monolayer wounds, by increasing the motility of the endothelial cells. Wound closure in HUVEC cells was partly inhibited by the antibodies against HGF. The stromal microenvironment regulates wound healing responses and often promotes tumorigenesis. Nemosis offers clues to the activation process of stromal fibroblasts and provides a model to study the part they play in angiogenesis-related conditions, as well as possibilities for therapeutical approaches desiring angiogenesis in tissue.

  14. Acrylamide induces accelerated endothelial aging in a human cell model.

    Science.gov (United States)

    Sellier, Cyril; Boulanger, Eric; Maladry, François; Tessier, Frédéric J; Lorenzi, Rodrigo; Nevière, Rémi; Desreumaux, Pierre; Beuscart, Jean-Baptiste; Puisieux, François; Grossin, Nicolas

    2015-09-01

    Acrylamide (AAM) has been recently discovered in food as a Maillard reaction product. AAM and glycidamide (GA), its metabolite, have been described as probably carcinogenic to humans. It is widely established that senescence and carcinogenicity are closely related. In vitro, endothelial aging is characterized by replicative senescence in which primary cells in culture lose their ability to divide. Our objective was to assess the effects of AAM and GA on human endothelial cell senescence. Human umbilical vein endothelial cells (HUVECs) cultured in vitro were used as model. HUVECs were cultured over 3 months with AAM or GA (1, 10 or 100 μM) until growth arrest. To analyze senescence, β-galactosidase activity and telomere length of HUVECs were measured by cytometry and semi-quantitative PCR, respectively. At all tested concentrations, AAM or GA reduced cell population doubling compared to the control condition (p < 0.001). β-galactosidase activity in endothelial cells was increased when exposed to AAM (≥10 μM) or GA (≥1 μM) (p < 0.05). AAM (≥10 μM) or GA (100 μM) accelerated telomere shortening in HUVECs (p < 0.05). In conclusion, in vitro chronic exposure to AAM or GA at low concentrations induces accelerated senescence. This result suggests that an exposure to AAM might contribute to endothelial aging.

  15. Characterization and comparison of embryonic stem cell-derived KDR+ cells with endothelial cells.

    Science.gov (United States)

    Sun, Xuan; Cheng, Lamei; Duan, Huaxin; Lin, Ge; Lu, Guangxiu

    2012-09-01

    Growing interest in utilizing endothelial cells (ECs) for therapeutic purposes has led to the exploration of human embryonic stem cells (hESCs) as a potential source for endothelial progenitors. In this study, ECs were induced from hESC lines and their biological characteristics were analyzed and compared with both cord blood endothelial progenitor cells (CBEPCs) and human umbilical vein endothelial cells (HUVECs) in vitro. The results showed that isolated embryonic KDR+ cells (EC-KDR+) display characteristics that were similar to CBEPCs and HUVECs. EC-KDR+, CBEPCs and HUVECs all expressed CD31 and CD144, incorporated DiI-Ac-LDL, bound UEA1 lectin, and were able to form tube-like structures on Matrigel. Compared with CBEPCs and HUVECs, the expression level of endothelial progenitor cell markers such as CD133 and KDR in EC-KDR+ was significantly higher, while the mature endothelial marker vWF was lowly expressed in EC-KDR+. In summary, the study showed that EC-KDR+ are primitive endothelial-like progenitors and might be a potential source for therapeutic vascular regeneration and tissue engineering.

  16. Effects of Granulocyte-Macrophage Colony-Stimulating (GM-CSF) Factor on Corneal Epithelial Cells in Corneal Wound Healing Model.

    Science.gov (United States)

    Rho, Chang Rae; Park, Mi-young; Kang, Seungbum

    2015-01-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that activates granulocyte and macrophage cell lineages. It is also known to have an important function in wound healing. This study investigated the effect of GM-CSF in wound healing of human corneal epithelial cells (HCECs). We used human GM-CSF derived from rice cells (rice cell-derived recombinant human GM-CSF; rhGM-CSF). An in vitro migration assay was performed to investigate the migration rate of HCECs treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml). MTT assay and flow cytometric analysis were used to evaluate the proliferative effect of rhGM-CSF. The protein level of p38MAPK was analyzed by western blotting. For in vivo analysis, 100 golden Syrian hamsters were divided into four groups, and their corneas were de-epithelialized with alcohol and a blade. The experimental groups were treated with 10, 20, or 50 μg/ml rhGM-CSF four times daily, and the control group was treated with phosphate-buffered saline. The corneal wound-healing rate was evaluated by fluorescein staining at the initial wounding and 12, 24, 36, and 48 hours after epithelial debridement. rhGM-CSF accelerated corneal epithelial wound healing both in vitro and in vivo. MTT assay and flow cytometric analysis revealed that rhGM-CSF treatment had no effects on HCEC proliferation. Western blot analysis demonstrated that the expression level of phosphorylated p38MAPK increased with rhGM-CSF treatment. These findings indicate that rhGM-CSF enhances corneal wound healing by accelerating cell migration.

  17. Effects of Granulocyte-Macrophage Colony-Stimulating (GM-CSF Factor on Corneal Epithelial Cells in Corneal Wound Healing Model.

    Directory of Open Access Journals (Sweden)

    Chang Rae Rho

    Full Text Available Granulocyte-macrophage colony-stimulating factor (GM-CSF is a pleiotropic cytokine that activates granulocyte and macrophage cell lineages. It is also known to have an important function in wound healing. This study investigated the effect of GM-CSF in wound healing of human corneal epithelial cells (HCECs. We used human GM-CSF derived from rice cells (rice cell-derived recombinant human GM-CSF; rhGM-CSF. An in vitro migration assay was performed to investigate the migration rate of HCECs treated with various concentrations of rhGM-CSF (0.1, 1.0, and 10.0 μg/ml. MTT assay and flow cytometric analysis were used to evaluate the proliferative effect of rhGM-CSF. The protein level of p38MAPK was analyzed by western blotting. For in vivo analysis, 100 golden Syrian hamsters were divided into four groups, and their corneas were de-epithelialized with alcohol and a blade. The experimental groups were treated with 10, 20, or 50 μg/ml rhGM-CSF four times daily, and the control group was treated with phosphate-buffered saline. The corneal wound-healing rate was evaluated by fluorescein staining at the initial wounding and 12, 24, 36, and 48 hours after epithelial debridement. rhGM-CSF accelerated corneal epithelial wound healing both in vitro and in vivo. MTT assay and flow cytometric analysis revealed that rhGM-CSF treatment had no effects on HCEC proliferation. Western blot analysis demonstrated that the expression level of phosphorylated p38MAPK increased with rhGM-CSF treatment. These findings indicate that rhGM-CSF enhances corneal wound healing by accelerating cell migration.

  18. Isolation and culture of human umbilical vein endothelial cells (HUVEC).

    Science.gov (United States)

    Cheung, Ambrose L

    2007-02-01

    Human-derived endothelial cells can now be routinely harvested from human umbilical veins. Studies with human umbilical vein endothelial cells (HUVEC) have been conducted with cells from passage 2 to 5. It is now also possible to cryopreserve primary and early-passaged HUVEC for future propagation and for forwarding to an end user by express courier. Stored HUVEC have been stably retrieved even after several years. These retrieval techniques have facilitated the deployment of HUVEC for many studies, including those for homeostasis, inflammatory disorders, atherosclerosis, cancer, and microbial adhesion and invasion. In this unit, we will delineate the procedure for harvesting, propagation, and storage of HUVEC.

  19. Corneal Laceration

    Medline Plus

    Full Text Available ... Laceration? Corneal Laceration Diagnosis Corneal Laceration Treatment What Is Corneal Laceration? Written By: Daniel Porter Reviewed By: ... A Harrison MD Sep. 01, 2016 The cornea is the clear front window of the eye . A ...

  20. Infection of hepatitis B virus in extrahepatic endothelial tissues mediated by endothelial progenitor cells

    Directory of Open Access Journals (Sweden)

    Zhang Lili

    2007-04-01

    Full Text Available Abstract Background Hepatitis B virus (HBV replication has been reported to be involved in many extrahepatic viral disorders; however, the mechanism by which HBV is trans-infected into extrahepatic tissues such as HBV associated myocarditis remains largely unknown. Results In this study, we showed that human cord blood endothelial progenitor cells (EPCs, but not human umbilical vein endothelial cells (HUVECs could be effectively infected by uptake of HBV in vitro. Exposure of EPCs with HBV resulted in HBV DNA and viral particles were detected in EPCs at day 3 after HBV challenge, which were peaked around day 7 and declined in 3 weeks. Consistently, HBV envelope surface and core antigens were first detected in EPCs at day 3 after virus challenge and were retained to be detectable for 3 weeks. In contrast, HBV covalently closed circular DNA was not detected in EPCs at any time after virus challenge. Intravenous transplantation of HBV-treated EPCs into myocardial infarction and acute renal ischemia mouse model resulted in incorporation of HBV into injured heart, lung, and renal capillary endothelial tissues. Conclusion These results strongly support that EPCs serve as virus carrier mediating HBV trans-infection into the injured endothelial tissues. The findings might provide a novel mechanism for HBV-associated myocarditis and other HBV-related extrahepatic diseases as well.

  1. Fibroblast growth factor receptor 2 (FGFR2) is required for corneal epithelial cell proliferation and differentiation during embryonic development.

    Science.gov (United States)

    Zhang, Jinglin; Upadhya, Dinesh; Lu, Lin; Reneker, Lixing W

    2015-01-01

    Fibroblast growth factors (FGFs) play important roles in many aspects of embryonic development. During eye development, the lens and corneal epithelium are derived from the same surface ectodermal tissue. FGF receptor (FGFR)-signaling is essential for lens cell differentiation and survival, but its role in corneal development has not been fully investigated. In this study, we examined the corneal defects in Fgfr2 conditional knockout mice in which Cre expression is activated at lens induction stage by Pax6 P0 promoter. The cornea in LeCre, Fgfr2(loxP/loxP) mice (referred as Fgfr2(CKO)) was analyzed to assess changes in cell proliferation, differentiation and survival. We found that Fgfr2(CKO) cornea was much thinner in epithelial and stromal layer when compared to WT cornea. At embryonic day 12.5-13.5 (E12.5-13.5) shortly after the lens vesicle detaches from the overlying surface ectoderm, cell proliferation (judged by labeling indices of Ki-67, BrdU and phospho-histone H3) was significantly reduced in corneal epithelium in Fgfr2(CKO) mice. At later stage, cell differentiation markers for corneal epithelium and underlying stromal mesenchyme, keratin-12 and keratocan respectively, were not expressed in Fgfr2(CKO) cornea. Furthermore, Pax6, a transcription factor essential for eye development, was not present in the Fgfr2(CKO) mutant corneal epithelial at E16.5 but was expressed normally at E12.5, suggesting that FGFR2-signaling is required for maintaining Pax6 expression in this tissue. Interestingly, the role of FGFR2 in corneal epithelial development is independent of ERK1/2-signaling. In contrast to the lens, FGFR2 is not required for cell survival in cornea. This study demonstrates for the first time that FGFR2 plays an essential role in controlling cell proliferation and differentiation, and maintaining Pax6 levels in corneal epithelium via ERK-independent pathways during embryonic development.

  2. Nanofiber density determines endothelial cell behavior on hydrogel matrix

    Energy Technology Data Exchange (ETDEWEB)

    Berti, Fernanda V., E-mail: fernanda@intelab.ufsc.br [Department of Chemical and Food Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Rambo, Carlos R. [Department of Electrical Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Dias, Paulo F. [Department of Cell Biology, Embryology and Genetics, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil); Porto, Luismar M. [Department of Chemical and Food Engineering, Federal University of Santa Catarina, 88040-900 Florianópolis, SC (Brazil)

    2013-12-01

    When cultured under static conditions, bacterial cellulose pellicles, by the nature of the polymer synthesis that involves molecular oxygen, are characterized by two distinct surface sides. The upper surface is denser in fibers (entangled) than the lower surface that shows greater surface porosity. Human umbilical vein endothelial cells (HUVECs) were used to exploit how the microarchitecture (i.e., surface porosity, fiber network structure, surface topology, and fiber density) of bacterial cellulose pellicle surfaces influence cell–biomaterial interaction and therefore cell behavior. Adhesion, cell ingrowth, proliferation, viability and cell death mechanisms were evaluated on the two pellicle surface sides. Cell behavior, including secondary necrosis, is influenced only by the microarchitecture of the surface, since the biomaterial is extremely pure (constituted of cellulose and water only). Cell–cellulose fiber interaction is the determinant signal in the cell–biomaterial responses, isolated from other frequently present interferences such as protein and other chemical traces usually present in cell culture matrices. Our results suggest that microarchitecture of hydrogel materials might determine the performance of biomedical products, such as bacterial cellulose tissue engineering constructs (BCTECs). - Highlights: • Topography of BC pellicle is relevant to determine endothelial cells' fate. • Cell–biomaterial response is affected by the topography of BC-pellicle surface. • Endothelial cells exhibit different behavior depending on the BC topography. • Apoptosis and necrosis of endothelial cells were affected by the BC topography.

  3. In vitro behaviour of endothelial cells on a titanium surface

    Directory of Open Access Journals (Sweden)

    Oliveira-Filho Ricardo

    2008-07-01

    Full Text Available Abstract Background Endothelial cells play an important role in the delivery of cells to the inflammation site, chemotaxis, cell adhesion and extravasation. Implantation of a foreign material into the human body determines inflammatory and repair reactions, involving different cell types with a plethora of released chemical mediators. The evaluation of the interaction of endothelial cells and implanted materials must take into account other parameters in addition to the analysis of maintenance of cell viability. Methods In the present investigation, we examined the behavior of human umbilical vein endothelial cells (HUVECs harvested on titanium (Ti, using histological and immunohistochemical methods. The cells, after two passages, were seeded in a standard density on commercially plate-shaped titanium pieces, and maintained for 1, 7 or 14 days. Results After 14 days, we could observe a confluent monolayer of endothelial cells (ECs on the titanium surface. Upon one-day Ti/cell contact the expression of fibronectin was predominantly cytoplasmatic and stronger than on the control surface. It was observed strong and uniform cell expression along the time of α5β1 integrin on the cells in contact with titanium. Conclusion The attachment of ECs on titanium was found to be related to cellular-derived fibronectin and the binding to its specific receptor, the α5β1 integrin. It was observed that titanium effectively serves as a suitable substrate for endothelial cell attachment, growth and proliferation. However, upon a 7-day contact with Ti, the Weibel-Palade bodies appeared to be not fully processed and exhibited an anomalous morphology, with corresponding alterations of PECAM-1 localization.

  4. Primary Corneal Squamous Cell Carcinoma in a Dog: Clinical and Histopathological Evaluation

    OpenAIRE

    Giovanni Barsotti; Lorenzo Ressel; Riccardo Finotello; Veronica Marchetti; Francesca Millanta

    2012-01-01

    An 8-year-old male pug with a 12-month history of a progressive nonpainful mass on the left cornea was evaluated. Ocular examination showed a severe bilateral keratoconjunctivitis sicca, pigmentary keratitis, and an exophytic irregular pink mass occupying approximately 75% of the total corneal surface of the left eye. A squamous cell carcinoma (SCC) was suspected on cytology, and clinical investigations showed no evidence of metastases. A transpalpebral enucleation was therefore performed, an...

  5. Endothelial Cells Stimulate Self-Renewal and Expand Neurogenesis of Neural Stem Cells

    Science.gov (United States)

    Shen, Qin; Goderie, Susan K.; Jin, Li; Karanth, Nithin; Sun, Yu; Abramova, Natalia; Vincent, Peter; Pumiglia, Kevin; Temple, Sally

    2004-05-01

    Neural stem cells are reported to lie in a vascular niche, but there is no direct evidence for a functional relationship between the stem cells and blood vessel component cells. We show that endothelial cells but not vascular smooth muscle cells release soluble factors that stimulate the self-renewal of neural stem cells, inhibit their differentiation, and enhance their neuron production. Both embryonic and adult neural stem cells respond, allowing extensive production of both projection neuron and interneuron types in vitro. Endothelial coculture stimulates neuroepithelial cell contact, activating Notch and Hes1 to promote self-renewal. These findings identify endothelial cells as a critical component of the neural stem cell niche.

  6. The SULFs, extracellular sulfatases for heparan sulfate, promote the migration of corneal epithelial cells during wound repair.

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    Inna Maltseva

    Full Text Available Corneal epithelial wound repair involves the migration of epithelial cells to cover the defect followed by the proliferation of the cells to restore thickness. Heparan sulfate proteoglycans (HSPGs are ubiquitous extracellular molecules that bind to a plethora of growth factors, cytokines, and morphogens and thereby regulate their signaling functions. Ligand binding by HS chains depends on the pattern of four sulfation modifications, one of which is 6-O-sulfation of glucosamine (6OS. SULF1 and SULF2 are highly homologous, extracellular endosulfatases, which post-synthetically edit the sulfation status of HS by removing 6OS from intact chains. The SULFs thereby modulate multiple signaling pathways including the augmentation of Wnt/ß-catenin signaling. We found that wounding of mouse corneal epithelium stimulated SULF1 expression in superficial epithelial cells proximal to the wound edge. Sulf1⁻/⁻, but not Sulf2⁻/⁻, mice, exhibited a marked delay in healing. Furthermore, corneal epithelial cells derived from Sulf1⁻/⁻ mice exhibited a reduced rate of migration in repair of a scratched monolayer compared to wild-type cells. In contrast, human primary corneal epithelial cells expressed SULF2, as did a human corneal epithelial cell line (THCE. Knockdown of SULF2 in THCE cells also slowed migration, which was restored by overexpression of either mouse SULF2 or human SULF1. The interchangeability of the two SULFs establishes their capacity for functional redundancy. Knockdown of SULF2 decreased Wnt/ß-catenin signaling in THCE cells. Extracellular antagonists of Wnt signaling reduced migration of THCE cells. However in SULF2- knockdown cells, these antagonists exerted no further effects on migration, consistent with the SULF functioning as an upstream regulator of Wnt signaling. Further understanding of the mechanistic action of the SULFs in promoting corneal repair may lead to new therapeutic approaches for the treatment of corneal injuries.

  7. Targeting brain microvascular endothelial cells: a therapeutic approach to neuroprotection against stroke

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    Qi-jin Yu

    2015-01-01

    Full Text Available Brain microvascular endothelial cells form the interface between nervous tissue and circulating blood, and regulate central nervous system homeostasis. Brain microvascular endothelial cells differ from peripheral endothelial cells with regards expression of specific ion transporters and receptors, and contain fewer fenestrations and pinocytotic vesicles. Brain microvascular endothelial cells also synthesize several factors that influence blood vessel function. This review describes the morphological characteristics and functions of brain microvascular endothelial cells, and summarizes current knowledge regarding changes in brain microvascular endothelial cells during stroke progression and therapies. Future studies should focus on identifying mechanisms underlying such changes and developing possible neuroprotective therapeutic interventions.

  8. Magnetizable stent-grafts enable endothelial cell capture

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    Tefft, Brandon J.; Uthamaraj, Susheil; Harburn, J. Jonathan; Hlinomaz, Ota; Lerman, Amir; Dragomir-Daescu, Dan; Sandhu, Gurpreet S.

    2017-04-01

    Emerging nanotechnologies have enabled the use of magnetic forces to guide the movement of magnetically-labeled cells, drugs, and other therapeutic agents. Endothelial cells labeled with superparamagnetic iron oxide nanoparticles (SPION) have previously been captured on the surface of magnetizable 2205 duplex stainless steel stents in a porcine coronary implantation model. Recently, we have coated these stents with electrospun polyurethane nanofibers to fabricate prototype stent-grafts. Facilitated endothelialization may help improve the healing of arteries treated with stent-grafts, reduce the risk of thrombosis and restenosis, and enable small-caliber applications. When placed in a SPION-labeled endothelial cell suspension in the presence of an external magnetic field, magnetized stent-grafts successfully captured cells to the surface regions adjacent to the stent struts. Implantation within the coronary circulation of pigs (n=13) followed immediately by SPION-labeled autologous endothelial cell delivery resulted in widely patent devices with a thin, uniform neointima and no signs of thrombosis or inflammation at 7 days. Furthermore, the magnetized stent-grafts successfully captured and retained SPION-labeled endothelial cells to select regions adjacent to stent struts and between stent struts, whereas the non-magnetized control stent-grafts did not. Early results with these prototype devices are encouraging and further refinements will be necessary in order to achieve more uniform cell capture and complete endothelialization. Once optimized, this approach may lead to more rapid and complete healing of vascular stent-grafts with a concomitant improvement in long-term device performance.

  9. Opioid-induced proliferation of vascular endothelial cells

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    Sandra Leo

    2009-05-01

    Full Text Available Sandra Leo1,2, Rony Nuydens1, Theo F Meert11Pain and Neurology, CNS Department, Johnson and Johnson Pharmaceutical Research and Development, a division of Janssen Pharmaceutica N.V, Beerse, Belgium; 2Laboratory of Biological Psychology, University of Leuven, Leuven, BelgiumAbstract: Angiogenesis is an important issue in cancer research and opioids are often used to treat pain in cancer patients. Therefore it is important to know if the use of opioids is associated with an aberrant stimulation of tumor growth triggered by the stimulation of angiogenesis in cancer patients. Some studies in the literature have suggested the presence of the μ3 opioid receptor, known as the receptor for many opioids, on endothelial cells, which are key players in the process of angiogenesis. In this study we used endothelial cells known to express the μ3 opioid receptor (MOR3, to evaluate the effects of morphine on angiogenesis. We first investigated the effect of morphine on the proliferation of endothelial cells. We showed that morphine is able to stimulate vascular endothelial cell proliferation in vitro. This effect of morphine is mediated by the mitogen-activated protein kinase (MAPK pathway as pre-treatment with PD98059 inhibited this excessive proliferation. Because previous studies indicated nitric oxide (NO as a downstream messenger we investigated the role of NO in the aberrant proliferation of endothelial cells. Our data could not confirm these findings using intracellular NO measurements and quantitative fluorescence microscopy. The potential use and pitfalls of opioids in cancer patients is discussed in light of these negative findings. Keywords: endothelial cells, morphine, cell proliferation, MAPK, nitric oxide, μ3 opioid receptor, angiogenesis

  10. Reproducibility of Corneal Graft Thickness measurements with COLGATE in patients who have undergone DSAEK (Descemet Stripping Automated Endothelial Keratoplasty

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    Wong Melissa HY

    2012-08-01

    Full Text Available Abstract Background The CorneaL GrAft Thickness Evaluation (COLGATE system was recently developed to facilitate the evaluation of corneal graft thickness from OCT images. Graft thickness measurement can be a surrogate indicator for detecting graft failure or success. The purpose of this study was to determine the reproducibility of the COLGATE system in measuring DSAEK graft area between two observers. Methods This was a prospective case series in which 50 anterior segment OCT images of patients who had undergone DSAEK in either eye were analysed. Two observers (MW, AC independently obtained the image analysis for the graft area using both semi automated and automated method. One week later, each observer repeated the analysis for the same set of images. Bland-Altman analysis was performed to analyze inter and intra observer agreement. Results There was strong intraobserver correlation between the 2 semi automated readings obtained by both observers. (r = 0.936 and r = 0.962. Intraobserver ICC for observer 1 was 0.936 (95% CI 0.890 to 0.963 and 0.967 (95% CI 0.942 to 0.981 for observer 2. Likewise, there was also strong interobserver correlation (r = 0.913 and r = 0.969. The interobserver ICC for the first measurements was 0.911 (95% CI 0.849 to 0.949 and 0.968 (95% CI 0.945 to 0.982 for the second. There was statistical difference between the automatic and the semi automated readings for both observers (p = 0.006, p = 0.003. The automatic readings gave consistently higher values than the semi automated readings especially in thin grafts. Conclusion The analysis from the COLGATE programme can be reproducible between different observers. Care must be taken when interpreting the automated analysis as they tend to over estimate measurements.

  11. Tumor endothelial cells express high pentraxin 3 levels.

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    Hida, Kyoko; Maishi, Nako; Kawamoto, Taisuke; Akiyama, Kosuke; Ohga, Noritaka; Hida, Yasuhiro; Yamada, Kenji; Hojo, Takayuki; Kikuchi, Hiroshi; Sato, Masumi; Torii, Chisaho; Shinohara, Nobuo; Shindoh, Masanobu

    2016-12-01

    It has been described that tumor progression has many similarities to inflammation and wound healing in terms of the signaling processes involved. Among biological responses, angiogenesis, which is necessary for tumor progression and metastasis, is a common hallmark; therefore, tumor blood vessels have been considered as important therapeutic targets in anticancer therapy. We focused on pentraxin 3 (PTX3), which is a marker of cancer-related inflammation, but we found no reports on its expression and function in tumor blood vessels. Here we showed that PTX3 is expressed in mouse and human tumor blood vessels based on immunohistochemical analysis. We found that PTX3 is upregulated in primary mouse and human tumor endothelial cells compared to normal endothelial cells. We also showed that PTX3 plays an important role in the proliferation of the tumor endothelial cells. These results suggest that PTX3 is an important target for antiangiogenic therapy.

  12. Corneal mucus plaques.

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    Fraunfelder, F T; Wright, P; Tripathi, R C

    1977-02-01

    Corneal mucus plaques adhered to the anterior corneal surface in 17 of 67 advanced cases of keratoconjunctivitis sicca. The plaques were translucent to opaque and varied in size and shape, from multiple isolated islands to bizarre patterns involving more than half the corneal surface. Ultrastructurally, they consisted of mucus mixed with desquamated degenerating epithelial cells and proteinaceous and lipoidal material. The condition may be symptomatic but can be controlled and prevented in most cases by topical ocular application of 10% acetylcysteine.

  13. Treponema pallidum Invades Intercellular Junctions of Endothelial Cell Monolayers

    Science.gov (United States)

    Thomas, D. Denee; Navab, Mahamad; Haake, David A.; Fogelman, Alan M.; Miller, James N.; Lovett, Michael A.

    1988-05-01

    The pathogenesis of syphilis reflects invasive properties of Treponema pallidum, but the actual mode of tissue invasion is unknown. We have found two in vitro parallels of treponemal invasiveness. We tested whether motile T. pallidum could invade host cells by determining the fate of radiolabeled motile organisms added to a HeLa cell monolayer; 26% of treponemes associated with the monolayer in a trypsin-resistant niche, presumably between the monolayer and the surface to which it adhered, but did not attain intracellularity. Attachment of T. pallidum to cultured human and rabbit aortic and human umbilical vein endothelial cells was 2-fold greater than to HeLa cells. We added T. pallidum to aortic endothelial cells grown on membrane filters under conditions in which tight intercellular junctions had formed. T. pallidum was able to pass through the endothelial cell monolayers without altering tight junctions, as measured by electrical resistance. In contrast, heat-killed T. pallidum and the nonpathogen Treponema phagedenis biotype Reiter failed to penetrate the monolayer. Transmission electron micrographs of sections of the monolayer showed T. pallidum in intercellular junctions. Our in vitro observations suggest that these highly motile spirochetes may leave the circulation by invading the junctions between endothelial cells.

  14. Effect of Antioxidants on Endothelial Cell Reactive Oxygen Species (ROI) Generation and Adhesion of Leukocytes to Endothelial Cells

    Institute of Scientific and Technical Information of China (English)

    Huang Qian; Michael Grafe; Kristoph Graf; Hans Lehmkuhl; Eckart Fleck

    2000-01-01

    Objective To investigate whether antioxidants inhibit adhesion of leukocytes to endothelium and furthermore, whether all antioxidants regulate NF-κB activation through a redox sensitive mechanism. Methods The effect of the antioxidative substances pyrrolidin dithiocarbamat (PDTC),dichloroisocumarin (DCI), chrysin and probucol on the endothelial leukocyte adhesion were examined under near physiological flow conditions. The antioxidative activity of antioxidants was measured in a DCF fluorescence assay with flow cytometry. The activation of NF-κB in endothelial cells was investigated in a gel shift assay. Results PDTC and probucol did not show an inhibitory effect to the formation of intracellular H2O2 in TNFct activated human vascular endothelial cells (HUVEC) . Chrysin showed a moderate effect.DCI showed a strong antioxidative effect. In contrast,PDTC and chrysin inhibited the adhesion of HL 60 cells to TNFa-stimulated HUVEC. DCI and probucol did not have influence on the adhesion within the area of the examined shear stresses. Only PDTC inhibited the TNFα-induced activation of NF-kB in endothelial cells.Conclusion The inhibition of the endothelial leukocyte adhesion by antioxidative substances is not to be explained by its antioxidative characteristics only. The inhibitory effect of PDTC on NF-kB activation was probably not related to its antioxidative properties.

  15. Arecoline inhibits endothelial cell growth and migration and the attachment to mononuclear cells

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    Shuei-Kuen Tseng

    2014-09-01

    Conclusion: Arecoline impaired vascular endothelial cells by inhibiting their growth and migration and their adhesion to U937 mononuclear cells. These results reveal that arecoline may contribute to the pathogenesis of oral submucous fibrosis and cardiovascular diseases by affecting endothelial cell function in BQ chewers.

  16. ROS, MAPK/ERK and PKC play distinct roles in EGF-stimulated human corneal cell proliferation and migration.

    Science.gov (United States)

    Huo, Y-N; Chen, W; Zheng, X-X

    2015-11-08

    Cornea is at the outermost surface of eye globe, and it easily receives damage from ultraviolet light exposure, physiology wounding, and infections. It is essential to understand the mechanisms controlling human corneal epithelial (HCE) cell proliferation and wound healing. Epidermal growth factor (EGF) could stimulate cell proliferation and migration in various cell types. Therefore, we investigated the roles and mechanisms of EGF on HCE cell proliferation and migration. CCK-8 kit and wound healing experiment were used to investigate HCE cell proliferation and cell migration, respectively. ROS activity was quantified by DCFDA and flow cytometry. Western blot and Q-PCR were performed to examine protein and RNA levels. EGF could promote HCE cell proliferation and migration in both physiology status and UV irradiation conditions, which is used to mimic the disease condition in human corneal epithelial cells. Interestingly, the promotion effect of EGF on HCE cell proliferation is mainly mediated by activated ROS signaling under disease condition. However, the EGF function is mediated by ROS and MAPK/ERK pathway in EGF-treated corneal epithelial cells in physiology status, in which ROS and MAPK/ERK pathway have no mutual influence on the other signaling pathway in EGF-stimulated corneal epithelial cells. We also revealed that MAPK/ERK pathway instead of ROS mediates EGF-stimulated HCE cell migration. Interestingly, we found that PKC proteins were downregulated by EGF in HCE cells that is partially mediated by ROS signaling, while PKC pathway was not involved in EGF-stimulated corneal cell proliferation and migration. EGF promotes human corneal cell proliferation and migration both in physiology and disease conditions, and ROS, MAPK/ERK and PKC pathways play different roles in these processes.

  17. Intravital two-photon microscopy of immune cell dynamics in corneal lymphatic vessels.

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    Philipp Steven

    Full Text Available BACKGROUND: The role of lymphatic vessels in tissue and organ transplantation as well as in tumor growth and metastasis has drawn great attention in recent years. METHODOLOGY/PRINCIPAL FINDINGS: We now developed a novel method using non-invasive two-photon microscopy to simultaneously visualize and track specifically stained lymphatic vessels and autofluorescent adjacent tissues such as collagen fibrils, blood vessels and immune cells in the mouse model of corneal neovascularization in vivo. The mouse cornea serves as an ideal tissue for this technique due to its easy accessibility and its inducible and modifiable state of pathological hem- and lymphvascularization. Neovascularization was induced by suture placement in corneas of Balb/C mice. Two weeks after treatment, lymphatic vessels were stained intravital by intrastromal injection of a fluorescently labeled LYVE-1 antibody and the corneas were evaluated in vivo by two-photon microscopy (TPM. Intravital TPM was performed at 710 nm and 826 nm excitation wavelengths to detect immunofluorescence and tissue autofluorescence using a custom made animal holder. Corneas were then harvested, fixed and analyzed by histology. Time lapse imaging demonstrated the first in vivo evidence of immune cell migration into lymphatic vessels and luminal transport of individual cells. Cells immigrated within 1-5.5 min into the vessel lumen. Mean velocities of intrastromal corneal immune cells were around 9 µm/min and therefore comparable to those of T-cells and macrophages in other mucosal surfaces. CONCLUSIONS: To our knowledge we here demonstrate for the first time the intravital real-time transmigration of immune cells into lymphatic vessels. Overall this study demonstrates the valuable use of intravital autofluorescence two-photon microscopy in the model of suture-induced corneal vascularizations to study interactions of immune and subsequently tumor cells with lymphatic vessels under close as possible

  18. Effects of irradiated biodegradable polymer in endothelial cell monolayer formation

    Energy Technology Data Exchange (ETDEWEB)

    Arbeitman, Claudia R.; Grosso, Mariela F. del [CONICET – Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina); Gerencia de Investigación y Aplicaciones, TANDAR-CNEA (Argentina); Behar, Moni [Instituto de Física, UFRGS, Porto Alegre, RS (Brazil); García Bermúdez, Gerardo, E-mail: ggb@tandar.cnea.gov.ar [CONICET – Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina); Gerencia de Investigación y Aplicaciones, TANDAR-CNEA (Argentina); Escuela de Ciencia y Tecnología, UNSAM (Argentina)

    2013-11-01

    In this work we study cell adhesion, proliferation and cell morphology of endothelial cell cultured on poly-L-lactide acid (PLLA) modified by heavy ion irradiation. Thin films of PLLA samples were irradiated with sulfur (S) at energies of 75 MeV and gold (Au) at 18 MeV ion-beams. Ion beams were provided by the Tandar (Buenos Aires, Argentina) and Tandetron (Porto Alegre, Brazil) accelerators, respectively. The growth of a monolayer of bovine aortic endothelial cells (BAEC) onto unirradiated and irradiated surfaces has been studied by in vitro techniques in static culture. Cell viability and proliferation increased on modified substrates. But the results on unirradiated samples, indicate cell death (necrosis/apoptosis) with the consequent decrease in proliferation. We analyzed the correlation between irradiation parameters and cell metabolism and morphology.

  19. Contractile proteins of endothelial cells, platelets and smooth muscle.

    Science.gov (United States)

    Becker, C G; Nachman, R L

    1973-04-01

    In experiments described herein it was observed, by direct and indirect immunofluorescence technics, that rabbit antisera to human platelet actomyosin (thrombosthenin) stained mature megakaryocytes, blood platelets, endothelial cells and smooth muscle cells of arteries and veins, endothelial cells of liver sinusoids and certain capillaries, uterine smooth muscle cells, myoepithelial cells, perineurial cells of peripheral nerves and "fibroblastic" cells of granulation tissue. The specificity of immunohistologic staining was confirmed by appropriate absorption and blocking studies and immunodiffusional analysis in agarose gel. It was also observed by immunodiffusional analysis in agarose gel, electrophoresis of actomyosin fragments in polyacrylamide gels, immune inhibition of actomyosin ATPase activity and immune aggregation of platelets that uterine and platelet actomyosin are partially, but not completely, identical.

  20. Selenium-binding lactoferrin is taken into corneal epithelial cells by a receptor and prevents corneal damage in dry eye model animals.

    Science.gov (United States)

    Higuchi, Akihiro; Inoue, Hiroyoshi; Kaneko, Yoshio; Oonishi, Erina; Tsubota, Kazuo

    2016-11-11

    The ocular surface is strongly affected by oxidative stress, which causes many ocular diseases including dry eye. Previously, we showed that selenium compounds, e.g., selenoprotein P and Se-lactoferrin, were candidates for treatment of dry eye. This paper shows the efficacy of Se-lactoferrin for the treatment of dry eye compared with Diquas as a control drug using two dry eye models and incorporation of lactoferrin into corneal epithelial cells via lactoferrin receptors. We show the efficacy of Se-lactoferrin eye drops in the tobacco smoke exposure rat dry eye model and short-term rabbit dry eye model, although Diquas eye drops were only effective in the short-term rabbit dry eye model. These results indicate that Se-lactoferrin was useful in the oxidative stress-causing dry eye model. Se-lactoferrin was taken into corneal epithelium cells via lactoferrin receptors. We identified LRP1 as the lactoferrin receptor in the corneal epithelium involved in lactoferrin uptake. Se-lactoferrin eye drops did not irritate the ocular surface of rabbits. Se-lactoferrin was an excellent candidate for treatment of dry eye, reducing oxidative stress by a novel mechanism.

  1. Graft stability after endothelial keratoplasty

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    Jovanović Vesna

    2015-01-01

    Full Text Available Bacground/Aim. Techniques for replacing the corneal endothelium have been improved. The host-graft interface is the key to graft adhesion and visual recovery. The aim of this study was to establish graft stability after Descemet stripping with endothelial keratoplasty (DSEK, compare it to the graft stability after endothelial keratoplasty with the intact posterior corneal layers (nDSEK in the rabbit cornea, and to investigate the nature of wound healing. Methods. Adult white rabbits (n = 20 were divided in two experimental groups: ten rabbits underwent monocular DSEK, and ten rabbits underwent endothelial keratoplasty without Descemet stripping (nDSEK. On the second postoperative day a horizontal dislocation of the graft was tried using the Lindstrom roller in each animal. Corneas were processed for the light microscopy study. Results. Rolling the Lindstrom instrument over the corneal surface did not cause horizontal dislocation in any of the operated eyes. In the DSEK group light microscopy revealed the lack of inflammation and fibrosis at the clearly distinctive donor-recipient interface (DRI. Retrocorneal membrane was found in two eyes. In nDSEK group, the host Descemet` s membrane (DM was intact without endothelial cells, with good graft apposition, without inflammation, fibrosis, or retrocorneal membrane. Conclusion. This study suggests that there is no difference in graft stability in DSEK compared to nDSEK in rabbit corneas. Wounds healed at DRI by hypocellular scarring only in both experimental groups.

  2. Endothelial progenitor cells induce a phenotype shift in differentiated endothelial cells towards PDGF/PDGFRβ axis-mediated angiogenesis.

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    Moritz Wyler von Ballmoos

    Full Text Available BACKGROUND: Endothelial Progenitor Cells (EPC support neovascularization and regeneration of injured endothelium both by providing a proliferative cell pool capable of differentiation into mature vascular endothelial cells and by secretion of angiogenic growth factors. OBJECTIVE: The aim of this study was to investigate the role of PDGF-BB and PDGFRβ in EPC-mediated angiogenesis of differentiated endothelial cells. METHODS AND RESULTS: Conditioned medium from human EPC (EPC-CM cultured in hypoxic conditions contained substantially higher levels of PDGF-BB as compared to normoxic conditions (P<0.01. EPC-CM increased proliferation (1.39-fold; P<0.001 and migration (2.13-fold; P<0.001 of isolated human umbilical vein endothelial cells (HUVEC, as well as sprouting of vascular structures from ex vivo cultured aortic rings (2.78-fold increase; P = 0.01. The capacity of EPC-CM to modulate the PDGFRβ expression in HUVEC was assessed by western blot and RT-PCR. All the pro-angiogenic effects of EPC-CM on HUVEC could be partially inhibited by inactivation of PDGFRβ (P<0.01. EPC-CM triggered a distinct up-regulation of PDGFRβ (2.5±0.5; P<0.05 and its phosphorylation (3.6±0.6; P<0.05 in HUVEC. This was not observed after exposure of HUVEC to recombinant human PDGF-BB alone. CONCLUSION: These data indicate that EPC-CM sensitize endothelial cells and induce a pro-angiogenic phenotype including the up-regulation of PDGFRβ, thereby turning the PDGF/PDGFRβ signaling-axis into a critical element of EPC-induced endothelial angiogenesis. This finding may be utilized to enhance EPC-based therapy of ischemic tissue in future.

  3. Downregulation of PTEN at Corneal Wound Sites Accelerates Wound Healing through Increased Cell Migration

    Science.gov (United States)

    Cao, Lin; Graue-Hernandez, Enrique O.; Tran, Vu; Reid, Brian; Pu, Jin; Mannis, Mark J.

    2011-01-01

    Purpose. The PI3K/Akt pathway is required for cell polarization and migration, whereas the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) has inhibitory effects on the PI3K/Akt pathway. The authors therefore hypothesized that wounding would downregulate PTEN and that this downregulation would enhance wound healing. Methods. In human corneal epithelial (HCE) cell monolayer and rat cornea scratch wound models, the authors investigated PTEN and Akt expression using Western blot and immunofluorescence analyses. The effects of PTEN and PI3K inhibitors dipotassium bisperoxo (picolinato) oxovanadate (bpv(pic)) and LY294002 on cell migration and wound closure were investigated using time-lapse imaging. Finally, the authors investigated the effect of PTEN inhibition on wound healing in whole rat eyes. Results. In HCE cell monolayer and rat cornea, PTEN was downregulated at the wound edges within 30 minutes of wounding. The downregulation of PTEN was causal in a simultaneous increase in Akt activation, which was responsible for a significant increase in individual cell migration rate from 8.8 μm/h to 17.3 μm/h. An increased migration rate was maintained for 20 hours. PTEN inhibition significantly enhanced the wound healing rate in the HCE cell monolayer from 10 minutes onward after treatment and reduced the healing time in eye organ culture from 30 to 20 hours. Conclusions. Injury to the corneal epithelium downregulates the expression of PTEN at wound edges, allowing increased PI3K/Akt signaling, thereby contributing to a significant enhancement of cell migration and wound healing. These results suggest that PTEN inhibition may be an effective treatment for corneal injury. PMID:21212174

  4. Ex Vivo Behaviour of Human Bone Tumor Endothelial Cells

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    Infante, Teresa [SDN-Foundation, Institute of Diagnostic and Nuclear Development, IRCCS, 80143 Naples (Italy); Cesario, Elena [Department of Biochemistry and Biophysics, Second University of Naples, 80138 Naples (Italy); Gallo, Michele; Fazioli, Flavio [Division of Skeletal Muscles Oncology Surgery, National Cancer Institute, Pascale Foundation, 80131 Naples (Italy); De Chiara, Annarosaria [Anatomic Pathology Unit, National Cancer Institute, Pascale Foundation, 80131 Naples (Italy); Tutucci, Cristina; Apice, Gaetano [Medical Oncology of Bone and Soft Sarcoma tissues Unit, National Cancer Institute, Pascale Foundation, 80131 Naples (Italy); Nigris, Filomena de, E-mail: filomena.denigris@unina2.it [Department of Biochemistry and Biophysics, Second University of Naples, 80138 Naples (Italy)

    2013-04-11

    Cooperation between endothelial cells and bone in bone remodelling is well established. In contrast, bone microvasculature supporting the growth of primary tumors and metastasis is poorly understood. Several antiangiogenic agents have recently been undergoing trials, although an extensive body of clinical data and experimental research have proved that angiogenic pathways differ in each tumor type and stage. Here, for the first time, we characterize at the molecular and functional level tumor endothelial cells from human bone sarcomas at different stages of disease and with different histotypes. We selected a CD31{sup +} subpopulation from biopsies that displayed the capability to grow as adherent cell lines without vascular endothelial growth factor (VEGF). Our findings show the existence in human primary bone sarcomas of highly proliferative endothelial cells expressing CD31, CD44, CD105, CD146 and CD90 markers. These cells are committed to develop capillary-like structures and colony formation units, and to produce nitric oxide. We believe that a better understanding of tumor vasculature could be a valid tool for the design of an efficacious antiangiogenic therapy as adjuvant treatment of sarcomas.

  5. Growth-limiting role of endothelial cells in endoderm development.

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    Sand, Fredrik Wolfhagen; Hörnblad, Andreas; Johansson, Jenny K; Lorén, Christina; Edsbagge, Josefina; Ståhlberg, Anders; Magenheim, Judith; Ilovich, Ohad; Mishani, Eyal; Dor, Yuval; Ahlgren, Ulf; Semb, Henrik

    2011-04-15

    Endoderm development is dependent on inductive signals from different structures in close vicinity, including the notochord, lateral plate mesoderm and endothelial cells. Recently, we demonstrated that a functional vascular system is necessary for proper pancreas development, and that sphingosine-1-phosphate (S1P) exhibits the traits of a blood vessel-derived molecule involved in early pancreas morphogenesis. To examine whether S1P(1)-signaling plays a more general role in endoderm development, S1P(1)-deficient mice were analyzed. S1P(1) ablation results in compromised growth of several foregut-derived organs, including the stomach, dorsal and ventral pancreas and liver. Within the developing pancreas the reduction in organ size was due to deficient proliferation of Pdx1(+) pancreatic progenitors, whereas endocrine cell differentiation was unaffected. Ablation of endothelial cells in vitro did not mimic the S1P(1) phenotype, instead, increased organ size and hyperbranching were observed. Consistent with a negative role for endothelial cells in endoderm organ expansion, excessive vasculature was discovered in S1P(1)-deficient embryos. Altogether, our results show that endothelial cell hyperplasia negatively influences organ development in several foregut-derived organs.

  6. Endothelial cells stimulate growth of normal and cancerous breast epithelial cells in 3D culture

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    Magnusson Magnus K

    2010-07-01

    Full Text Available Abstract Background Epithelial-stromal interaction provides regulatory signals that maintain correct histoarchitecture and homeostasis in the normal breast and facilitates tumor progression in breast cancer. However, research on the regulatory role of the endothelial component in the normal and malignant breast gland has largely been neglected. The aim of the study was to investigate the effects of endothelial cells on growth and differentiation of human breast epithelial cells in a three-dimensional (3D co-culture assay. Methods Breast luminal and myoepithelial cells and endothelial cells were isolated from reduction mammoplasties. Primary cells and established normal and malignant breast cell lines were embedded in reconstituted basement membrane in direct co-culture with endothelial cells and by separation of Transwell filters. Morphogenic and phenotypic profiles of co-cultures was evaluated by phase contrast microscopy, immunostaining and confocal microscopy. Results In co-culture, endothelial cells stimulate proliferation of both luminal- and myoepithelial cells. Furthermore, endothelial cells induce a subpopulation of luminal epithelial cells to form large acini/ducts with a large and clear lumen. Endothelial cells also stimulate growth and cloning efficiency of normal and malignant breast epithelial cell lines. Transwell and gradient co-culture studies show that endothelial derived effects are mediated - at least partially - by soluble factors. Conclusion Breast endothelial cells - beside their role in transporting nutrients and oxygen to tissues - are vital component of the epithelial microenvironment in the breast and provide proliferative signals to the normal and malignant breast epithelium. These growth promoting effects of endothelial cells should be taken into consideration in breast cancer biology.

  7. Endothelial progenitor cell differentiation using cryopreserved, umbilical cord blood-derived mononuclear cells

    Institute of Scientific and Technical Information of China (English)

    Jun-ho JANG; Hugh C KIM; Sun-kyung KIM; Jeong-eun CHOI; Young-jin KIM; Hyun-woo LEE; Seok-yun KANG; Joon-seong PARK; Jin-hyuk CHOI; Ho-yeong LIM

    2007-01-01

    Aim: To investigate the endothelial differentiation potentiality of umbilical cord blood (UCB), we induced the differentiation of endothelial progenitor cells (EPC)from cryopreserved UCB-derived mononuclear cells (MNC). Methods: MNC from cryopreserved UCB and peripheral blood (PB) were cultured in M199 medium with endothelial cell growth supplements for 14 d. EPC were characterized by RT-PCR,flow cytometry, and immunocytochemistry analysis. The proliferation of differen-tiated EPC was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTI') assay, and vascular endothelial growth factor (VEGF) concentra-tion was measured using an ELISA kit. Characteristics of UCB-derived EPC were compared with those of PB-derived EPC. Results: A number of round-shaped cells were loosely attached to the bottom after 24 h culture, and numerous spindle-shaped cells began to appear from the round-shaped ones on d 7. Those cells expressed endothelial markers such as, Fit-1/VEGFR-1, ecNOS, VE-cadherin, yon Willebrand factor, and secreted VEGF. The patterns of endothelial markers of EPC from PB and UCB did not show striking differences. The results of the prolifera-tion and secretion of VEGF were also similar. Conclusion: We successfully cul-tured UCB cells stored at -196 ℃ into cells with the quality of endothelial cells.Those EPC could be used for angiogenic therapeutics by activating adjacent endothelial cells and enhancing angiogenesis.

  8. Biomechanical changes in endothelial cells result from an inflammatory response

    Science.gov (United States)

    Vaitkus, Janina; Stroka, Kimberly; Aranda-Espinoza, Helim

    2012-02-01

    During periods of infection and disease, the immune system induces the release of TNF-α, an inflammatory cytokine, from a variety of cell types, such as macrophages. TNF-α, while circulating in the vasculature, binds to the apical surface of endothelial cells and causes a wide range of biological and mechanical changes to the endothelium. While the biological changes have been widely studied, the biomechanical aspects have been largely unexplored. Here, we investigated the biomechanical changes of the endothelium as a function of TNF-α treatment. First, we studied the traction forces applied by the endothelium, an effect that is much less studied than others. Through the use of traction force microscopy, we found that TNF-α causes an increase in traction forces applied by the endothelial cells as compared to non-treated cells. Then, we investigated cell morphology, cell mechanics, migration, and cytoskeletal dynamics. We found that in addition to increasing applied traction forces, TNF-α causes an increase in cell area and aspect ratio on average, as well as a shift in the organization of F-actin filaments within the cell. Combining these findings together, our results show that an inflammatory response heavily impacts the morphology, cell mechanics, migration, cytoskeletal dynamics, and applied traction forces of endothelial cells.

  9. Endothelial Cells Stimulate Self-Renewal and Expand Neurogenesis of Neural Stem Cells

    National Research Council Canada - National Science Library

    Qin Shen; Susan K. Goderie; Li Jin; Nithin Karanth; Yu Sun; Natalia Abramova; Peter Vincent; Kevin Pumiglia; Sally Temple

    2004-01-01

    .... We show that endothelial cells but not vascular smooth muscle cells release soluble factors that stimulate the self-renewal of neural stem cells, inhibit their differentiation, and enhance their neuron production...

  10. Gr-1intCD11b+ myeloid-derived suppressor cells accumulate in corneal allograft and improve corneal allograft survival.

    Science.gov (United States)

    Choi, Wungrak; Ji, Yong Woo; Ham, Hwa-Yong; Yeo, Areum; Noh, Hyemi; Jin, Su-Eon; Song, Jong Suk; Kim, Hyeon Chang; Kim, Eung Kwon; Lee, Hyung Keun

    2016-12-01

    We identified the characteristics of myeloid-derived suppressor cells (MDSCs) and investigated their mechanism of induction and their functional role in allograft rejection using a murine corneal allograft model. In mice, MDSCs coexpress CD11b and myeloid differentiation antigen Gr-1. Gr-1(+)CD11b(+) cells infiltrated allografted corneas between 4 d and 4 wk after surgery; however, the frequencies of Gr-1(+)CD11b(+) cells were not different between accepted and rejected allografts or in peripheral blood or BM. Of interest, Gr-1(int)CD11b(+) cells, but not Gr-1(hi)CD11b(+) cells, infiltrated the accepted graft early after surgery and expressed high levels of immunosuppressive cytokines, including IL-10, TGF-β, and TNF-related apoptosis-inducing ligand. This population remained until 4 wk after surgery. In vitro, only high dose (>100 ng/ml) of IFN-γ plus GM-CSF could induce immunosuppressive cytokine expression in Gr-1(int)CD11b(+) cells. Furthermore, adoptive transfer of Gr-1(int)CD11b(+) cells reduced T cell infiltration, which improved graft survival. In conclusion, high-dose IFN-γ in allograft areas is essential for development of Gr-1(int)CD11b(+) MDSCs in corneal allografts, and subtle environmental changes in the early period of the allograft can result in a large difference in graft survival.

  11. High glucose mediates endothelial-to-chondrocyte transition in human aortic endothelial cells

    Directory of Open Access Journals (Sweden)

    Tang Rining

    2012-09-01

    Full Text Available Abstract Background Vascular calcification is one of the common complications in diabetes mellitus. Many studies have shown that high glucose (HG caused cardiovascular calcification, but its underlying mechanism is not fully understood. Recently, medial calcification has been most commonly described in the vessels of patients with diabetes. Chondrocytes were involved in the medial calcification. Recent studies have shown that the conversion into mesenchymal stem cells (MSCs via the endothelial-to-mesenchymal transition (EndMT could be triggered in chondrocytes. Our previous research has indicated that HG induced EndMT in human aortic endothelial cells (HAECs. Therefore, we addressed the question of whether HG-induced EndMT could be transitioned into MSCs and differentiated into chondrocytes. Methods HAECs were divided into three groups: a normal glucose (NG group, HG group (30 mmol/L, and mannitol (5.5 mmol/L NG + 24.5 mmol/L group. Pathological changes were investigated using fluorescence microscopy and electron microscopy. Immunofluorescence staining was performed to detect the co-expression of endothelial markers, such as CD31, and fibroblast markers, such as fibroblast-specific protein 1 (FSP-1. The expression of FSP-1 was detected by real time-PCR and western blots. Endothelial-derived MSCs were grown in MSC medium for one week. The expression of the MSCs markers STRO-1, CD44, CD10 and the chondrocyte marker SOX9 was detected by immunofluorescence staining and western blots. Chondrocyte expression was detected by alcian blue staining. Calcium deposits were analyzed by alizarin red staining. Results The incubation of HAECs exposed to HG resulted in a fibroblast-like phenotype. Double staining of the HAECs indicated a co-localization of CD31 and FSP-1. The expression of FSP-1 was significantly increased in the HG group, and the cells undergoing EndMT also expressed STRO-1, CD44 and SOX9 compared with the controls (P  Conclusions Our

  12. 14-3-3{sigma} controls corneal epithelial cell proliferation and differentiation through the Notch signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Xin, Ying [Stem Cell Institute, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Lu, Qingxian [Tumor Immunobiology Group, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Li, Qiutang, E-mail: q.li@louisville.edu [Stem Cell Institute, James Brown Cancer Center, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States); Department of Ophthalmology and Visual Sciences, University of Louisville School of Medicine, 301 E. Muhammad Ali Blvd., Louisville, KY 40202 (United States)

    2010-02-19

    14-3-3{sigma} (also called stratifin) is specifically expressed in the stratified squamous epithelium and its function was recently shown to be linked to epidermal stratification and differentiation in the skin. In this study, we investigated its role in corneal epithelium cell proliferation and differentiation. We showed that the 14-3-3{sigma} mutation in repeated epilation (Er) mutant mice results in a dominant negative truncated protein. Primary corneal epithelial cells expressing the dominant negative protein failed to undergo high calcium-induced cell cycle arrest and differentiation. We further demonstrated that blocking endogenous 14-3-3{sigma} activity in corneal epithelial cells by overexpressing dominative negative 14-3-3{sigma} led to reduced Notch activity and Notch1/2 transcription. Significantly, expression of the active Notch intracellular domain overcame the block in epithelial cell differentiation in 14-3-3{sigma} mutant-expressing corneal epithelial cells. We conclude that 14-3-3{sigma} is critical for regulating corneal epithelial proliferation and differentiation by regulating Notch signaling activity.

  13. Telmisartan Activates Endothelial Nitric Oxide Synthase via Ser1177 Phosphorylation in Vascular Endothelial Cells

    Science.gov (United States)

    Myojo, Masahiro; Nagata, Daisuke; Fujita, Daishi; Kiyosue, Arihiro; Takahashi, Masao; Satonaka, Hiroshi; Morishita, Yoshiyuki; Akimoto, Tetsu; Nagai, Ryozo; Komuro, Issei; Hirata, Yasunobu

    2014-01-01

    Because endothelial nitric oxide synthase (eNOS) has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177) in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172) and eNOS and the concentration of intracellular guanosine 3′,5′-cyclic monophosphate (cGMP). Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK) inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling. PMID:24827148

  14. Telmisartan activates endothelial nitric oxide synthase via Ser1177 phosphorylation in vascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Masahiro Myojo

    Full Text Available Because endothelial nitric oxide synthase (eNOS has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177 in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172 and eNOS and the concentration of intracellular guanosine 3',5'-cyclic monophosphate (cGMP. Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.

  15. High glucose augments stress-induced apoptosis in endothelial cells

    Institute of Scientific and Technical Information of China (English)

    Wenwen Zhong; Yang Liu; Hui Tian

    2009-01-01

    Hyperglycemia has been identified as one of the important factors involved in the microvascular complications of diabetes, and has been related to increased cardiovascular mortality. Endothelial damage and dysfunction result from diabetes; therefore, the aim of this study was to determine the response of endothelial cells to stressful stimuli, modelled in normal and high glucose concentrations in vitro. Eahy 926 endothelial cells were cultured in 5 mmol/L or 30 mmol/L glucose conditions for a 24 hour period and oxidative stress was induced by exposure to hydrogen peroxide (H2O2) or tumour necrosis factor- α (TNF- α ), following which the protective effect of the glucocorticoid dexamethasone was assessed. Apoptosis, necrosis and cell viability were determined using an ELISA for DNA fragmentation, an enzymatic lactate dehydrogenase assay and an MTT assay, respectively. High glucose significantly increased the susceptibility of Eahy 926 cells to apoptosis in the presence of 500 μmol/L H2O2, above that induced in normal glucose (P<0.02). A reduction of H2O2- and TNF- α -induced apoptosis occurred in both high and low glucose after treatment with dexametha-sone (P<0.05). Conclusion high glucose is effective in significantly augmenting stress caused by H2O2, but not in causing stress alone. These findings suggest a mechanism by which short term hyperglycemia may facilitate and augment endothelial damage.

  16. Endothelial progenitor cell-based neovascularization : implications for therapy

    NARCIS (Netherlands)

    Krenning, Guido; van Luyn, Marja J. A.; Harmsen, Martin C.

    2009-01-01

    Ischemic cardiovascular events are a major cause of death globally. Endothelial progenitor cell (EPC)-based approaches can result in improvement of vascular perfusion and might offer clinical benefit. However, although functional improvement is observed, the lack of long-term engraftment of EPCs int

  17. Endothelial cell density after deep anterior lamellar keratoplasty (Melles technique)

    NARCIS (Netherlands)

    Van Dooren, BTH; Mulder, PGH; Nieuwendaal, CP; Beekhuis, WH; Melles, GRJ

    PURPOSE: To measure the recipient endothelial cell loss after the Melles technique for deep anterior lamellar keratoplasty. METHODS: In 21 eyes of 21 patients, a deep anterior lamellar keratoplasty procedure was performed. Before surgery and at 6, 12, and 24 months after surgery, specular microscopy

  18. METABOLIC CAPACITY REGULATES IRON HOMEOSTATIS IN ENDOTHELIAL CELLS

    Science.gov (United States)

    The sensitivity of endothelial cells to oxidative stress and the high concentrations of iron in mitochondria led us to test the hypotheses that (1) changes in respiratory capacity alter iron homeostasis, and (2) lack of aerobic metabolism decreases labile iron stores and attenuat...

  19. Are endothelial cell bioeffects from acoustic droplet vaporization proximity dependent?

    Science.gov (United States)

    Seda, Robinson; Li, David; Fowlkes, J. Brian; Bull, Joseph

    2013-11-01

    Acoustic droplet vaporization (ADV) produces gas microbubbles that provide a means of selective occlusion in gas embolotherapy. Vaporization and subsequent occlusion occur inside blood vessels supplying the targeted tissue, such as tumors. Theoretical and computational studies showed that ADV within a vessel can impart high fluid mechanical stresses on the vessel wall. Previous in vitro studies have demonstrated that vaporization at an endothelial layer may affect cell attachment and viability. The current study is aimed at investigating the role of vaporization distance away from the endothelial layer. HUVECs were cultured in OptiCell™ chambers until reaching confluence. Dodecafluoropentane microdroplets were added, attaining a 10:1 droplet to cell ratio. A single ultrasound pulse (7.5 MHz) consisting of 16 cycles (~ 2 μs) and a 5 MPa peak rarefactional pressure was used to produce ADV while varying the vaporization distance from the endothelial layer (0 μm, 500 μm, 1000 μm). Results indicated that cell attachment and viability was significantly different if the distance was 0 μm (at the endothelial layer). Other distances were not significantly different from the control. ADV will significantly affect the endothelium if droplets are in direct contact with the cells. Droplet concentration and flow conditions inside blood vessels may play an important role. This work was supported by NIH grant R01EB006476.

  20. Effects of hypergravity on the angiogenic potential of endothelial cells

    NARCIS (Netherlands)

    Costa-Almeida, R. (Raquel); Carvalho, D.T.O. (Daniel T.O.); Ferreira, M.J.S. (Miguel J.S.); Aresta, G. (Guilherme); Gomes, M.E. (Manuela E.); Van Loon, J.J.W.A. (Jack J.W.A.); K. van der Heiden (Kim); Granja, P.L. (Pedro L.)

    2016-01-01

    textabstractAngiogenesis, the formation of blood vessels from pre-existing ones, is a key event in pathology, including cancer progression, but also in homeostasis and regeneration. As the phenotype of endothelial cells (ECs) is continuously regulated by local biomechanical forces, studying endothel

  1. Effect of propionyl-L-carnitine on human endothelial cells

    NARCIS (Netherlands)

    Hinsbergh, V.W.M. van; Scheffer, M.A.

    1991-01-01

    A possible protective effect of propionyl-L-carnitine on human endothelial cells was studied both under basal culture conditions and in the presence of agents capable of influencing oxidative damage, such as glucose/glucose oxidase and oxidized low-density lipoproteins. Propionyl-L-carnitine had no

  2. Nanoparticle accumulation and transcytosis in brain endothelial cell layers

    NARCIS (Netherlands)

    Ye, Dong; Raghnaill, Michelle Nic; Bramini, Mattia; Mahon, Eugene; Åberg, Christoffer; Salvati, Anna; Dawson, Kenneth A

    2013-01-01

    The blood-brain barrier (BBB) is a selective barrier, which controls and limits access to the central nervous system (CNS). The selectivity of the BBB relies on specialized characteristics of the endothelial cells that line the microvasculature, including the expression of intercellular tight juncti

  3. Bradykinin-mediated cell proliferation depends on transactivation of EGF receptor in corneal fibroblasts.

    Science.gov (United States)

    Cheng, Ching-Yi; Tseng, Hui-Ching; Yang, Chuen-Mao

    2012-04-01

    In previous studies, bradykinin (BK) has been shown to induce cell proliferation through BK B2 receptor (B2R) via p42/p44 MAPK in Statens Seruminstitut Rabbit Corneal Cells (SIRCs). In addition to this pathway, EGFR transactivation pathway has been implicated in linking a variety of G-protein coupled receptors to MAPK cascades. Here, we further investigate whether these transactivation mechanisms participating in BK-induced cell proliferation in SIRCs. Using an immunofluorescence staining and RT-PCR, we initially characterize that SIRCs were corneal fibroblasts and predominantly expressed B2R by BK. Inhibition of p42/p44 MAPK by the inhibitors of Src, EGFR, and Akt or transfection with respective siRNAs prevents BK-induced DNA synthesis in SIRCs. The mechanisms underlying these responses were mediated through phosphorylation of Src and EGFR via the formation of Src/EGFR complex which was attenuated by PP1 and AG1478. Moreover, BK-induced p42/p44 MAPK and Akt activation was mediated through EGFR transactivation, which was diminished by the inhibitors of MMP-2/9 and heparin-binding EGF-like factor (HB-EGF). Finally, increased nuclear translocation of Akt and p42/p44 MAPK turns on early gene expression leading to cell proliferation. These results suggest that BK-induced cell proliferation is mediated through c-Src-dependent transactivation of EGFR via MMP2/9-dependent pro-HB-EGF shedding linking to activation of Akt and p42/p44 MAPK in corneal fibroblasts. Copyright © 2011 Wiley Periodicals, Inc.

  4. The human corneal endothelium in keratoconus: A specular microscopic study.

    Science.gov (United States)

    Laing, R A; Sandstrom, M M; Berrospi, A R; Leibowitz, H M

    1979-10-01

    The corneal endothelium in 12 cases of keratoconus was examined with the clinical specular microscope. There appeared to be an increase in cellular pleomorphism with many cells considerably smaller than normal distributed throughout the endothelial cell population. There were also many large, elongated cells whose long axis showed a definite tendency to assume a similar directional orientation. The long axis of these cells seemed oriented toward the apex of the cone, and the cells appeared to have been stretched by the ectatic process. Many endothelial cells contained dark intracellular structures. Their significance is unknown. The single cornea in this series with a history of acute hydrops contained a localized area in which the endothelial cells were seven to ten times larger than normal. This suggests that rupture of the endothelium and Descemet's membrane, responsible for the acute edematous process, occurs at this site, and that the adjacent cells enlarged to fill the defect.

  5. Influence of extracellular matrix proteins and substratum topography on corneal epithelial cell alignment and migration.

    Science.gov (United States)

    Raghunathan, Vijaykrishna; McKee, Clayton; Cheung, Wai; Naik, Rachel; Nealey, Paul F; Russell, Paul; Murphy, Christopher J

    2013-08-01

    The basement membrane (BM) of the corneal epithelium presents biophysical cues in the form of topography and compliance that can impact the phenotype and behaviors of cells and their nuclei through modulation of cytoskeletal dynamics. In addition, it is also well known that the intrinsic biochemical attributes of BMs can modulate cell behaviors. In this study, the influence of the combination of exogenous coating of extracellular matrix proteins (ECM) (fibronectin-collagen [FNC]) with substratum topography was investigated on cytoskeletal architecture as well as alignment and migration of immortalized corneal epithelial cells. In the absence of FNC coating, a significantly greater percentage of cells aligned parallel with the long axis of the underlying anisotropically ordered topographic features; however, their ability to migrate was impaired. Additionally, changes in the surface area, elongation, and orientation of cytoskeletal elements were differentially influenced by the presence or absence of FNC. These results suggest that the effects of topographic cues on cells are modulated by the presence of surface-associated ECM proteins. These findings have relevance to experiments using cell cultureware with biomimetic biophysical attributes as well as the integration of biophysical cues in tissue-engineering strategies and the development of improved prosthetics.

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  1. File list: His.CDV.20.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  2. File list: DNS.CDV.50.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

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  3. File list: His.CDV.50.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

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  4. File list: Oth.CDV.05.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

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  5. File list: Oth.CDV.10.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

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  6. File list: Pol.CDV.10.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

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  7. File list: DNS.CDV.05.AllAg.Brachiocephalic_endothelial_cells [Chip-atlas[Archive

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  8. Mitochondrial dysfunction and oxidative stress in corneal disease.

    Science.gov (United States)

    Vallabh, Neeru A; Romano, Vito; Willoughby, Colin E

    2017-05-23

    The cornea is the anterior transparent surface and the main refracting structure of the eye. Mitochondrial dysfunction and oxidative stress are implicated in the pathogenesis of inherited (e.g. Kearns Sayre Syndrome) and acquired corneal diseases (e.g. keratoconus and Fuchs endothelial corneal dystrophy). Both antioxidants and reactive oxygen species are found in the healthy cornea. There is increasing evidence of imbalance in the oxidative balance and mitochondrial function in the cornea in disease states. The cornea is vulnerable to mitochondrial dysfunction and oxidative stress due to its highly exposed position to ultraviolet radiation and high oxygen tension. The corneal endothelium is vulnerable to accumulating mitochondrial DNA (mtDNA) damage due to the post- mitotic nature of endothelial cells, yet their mitochondrial genome is continually replicating and mtDNA mutations can develop and accumulate with age. The unique physiology of the cornea predisposes this structure to oxidative damage, and there is interplay between inherited and acquired mitochondrial dysfunction, oxidative damage and a number of corneal diseases. By targeting mitochondrial dysfunction in corneal disease, emerging treatments may prevent or reduce visual loss. Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

  9. Vascular endothelial cells and dysfunctions: role of melatonin.

    Science.gov (United States)

    Rodella, Luigi Fabrizio; Favero, Gaia; Foglio, Eleonora; Rossini, Claudia; Castrezzati, Stefania; Lonati, Claudio; Rezzani, Rita

    2013-01-01

    Several pathological conditions, including hypertension, atherosclerosis, diabetes, ischemia/reperfusion injury and nicotine-induced vasculopathy, are associated with vascular endothelial dysfunction characterized by altered secretory output of endothelial cells. Therefore there is a search for molecules and interventions that could restore endothelial function, in particular augmenting NO production, reducing the generation of free radicals and vasoconstrictors and preventing undesired inflammation. The pineal hormone melatonin exhibits several endothelium protective properties: it scavenges free radicals, activates antioxidant defence enzymes, normalizes lipid and blood pressure profile and increases NO bioavailability. Melatonin improved vascular function in experimental hypertension, reducing intimal infiltration and restoring NO production. Melatonin improved the NO pathway also in animal models for the study of diabetes and prevented NO down-regulation and adhesive molecules up-regulation in nicotine-induced vasculopathy. The protection against endothelial damage, vasoconstriction, platelet aggregation and leukocyte infiltration might contribute to the beneficial effects against ischemia-reperfusion injury by melatonin. Therefore, melatonin administration has endothelium-protective potential in several pathological conditions. Nevertheless, it still needs to be established, whether melatonin is able to revert already established endothelial dysfunction in these conditions.

  10. Molecular evidence and functional expression of multidrug resistance associated protein (MRP) in rabbit corneal epithelial cells.

    Science.gov (United States)

    Karla, Pradeep K; Pal, Dananjay; Mitra, Ashim K

    2007-01-01

    Multidrug resistance associated protein (MRP) is a major family of efflux transporters involved in drug efflux leading to drug resistance. The objective of this study was to explore physical barriers for ocular drug absorption and to verify if the role of efflux transporters. MRP-2 is a major homologue of MRP family and found to express on the apical side of cell membrane. Cultured Rabbit Corneal Epithelial Cells (rCEC) were selected as an in vitro model for corneal epithelium. [14C]-erythromycin which is a proven substrate for MRP-2 was selected as a model drug for functional expression studies. MK-571, a known specific and potent inhibitor for MRP-2 was added to inhibit MRP mediated efflux. Membrane fraction of rCEC was used for western blot analysis. Polarized transport of [14C]-erythromycin was observed in rCEC and transport from B-->A was significantly high than from A-->B. Permeability's increased significantly from A-->B in the presence of MK-571 and ketoconozole. Uptake of [14C]-erythromycin in the presence of MK-571 was significantly higher than control in rCEC. RT-PCR analysis indicated a unique and distinct band at approximately 498 bp corresponding to MRP-2 in rCEC and MDCK11-MRP-2 cells. Immunoprecipitation followed by Western Blot analysis indicated a specific band at approximately 190 kDa in membrane fraction of rCEC and MDCK11-MRP-2 cells. For the first time we have demonstrated high expression of MRP-2 in rabbit corneal epithelium and its functional activity causing drug efflux. RT-PCR, immunoprecipitation followed by Western blot analysis further confirms the result.

  11. Biomimetic stochastic topography and electric fields synergistically enhance directional migration of corneal epithelial cells in a MMP-3-dependent manner.

    Science.gov (United States)

    Gao, Jing; Raghunathan, Vijay Krishna; Reid, Brian; Wei, Dongguang; Diaz, Rodney C; Russell, Paul; Murphy, Christopher J; Zhao, Min

    2015-01-01

    Directed migration of corneal epithelial cells (CECs) is critical for maintenance of corneal homeostasis as well as wound healing. Soluble cytoactive factors and the intrinsic chemical attributes of the underlying extracellular matrix (ECM) participate in stimulating and directing migration. The central importance of the intrinsic biophysical attributes of the microenvironment of the cell in modulating an array of fundamental epithelial behaviors including migration has been widely documented. Among the best measures of these attributes are the intrinsic topography and stiffness of the ECM and electric fields (EFs). How cells integrate these multiple simultaneous inputs is not well understood. Here, we present a method that combines the use of (i) topographically patterned substrates (mean pore diameter 800nm) possessing features that approximate those found in the native corneal basement membrane; and (ii) EFs (0-150mVmm(-1)) mimicking those at corneal epithelial wounds that the cells experience in vivo. We found that topographic cues and EFs synergistically regulated directional migration of human CECs and that this was associated with upregulation of matrix metalloproteinase-3 (MMP3). MMP3 expression and activity were significantly elevated with 150mVmm(-1) applied-EF while MMP2/9 remained unaltered. MMP3 expression was elevated in cells cultured on patterned surfaces against planar surfaces. The highest single-cell migration rate was observed with 150mVmm(-1) applied EF on patterned and planar surfaces. When cultured as a confluent sheet, EFs induced collective cell migration on stochastically patterned surfaces compared with dissociated single-cell migration on planar surfaces. These results suggest significant interaction of biophysical cues in regulating cell behaviors and will help define design parameters for corneal prosthetics and help to better understand corneal wound healing.

  12. Effect of Excessive Potassium Iodide on Rat Aorta Endothelial Cells.

    Science.gov (United States)

    Zhang, Man; Zou, Xiaoyan; Lin, Xinying; Bian, Jianchao; Meng, Huicui; Liu, Dan

    2015-08-01

    The aim of the current study was to investigate the effect of excess iodine on rat aorta endothelial cells and the potential underlying mechanisms. Rat aorta endothelial cells were cultured with iodide ion (3506, 4076, 4647, 5218, 5789, 6360, 6931, and 7512 mg/L) for 48 h. Morphological changes of cells were observed with microscope after Wright-Giemsa staining and acridine orange staining. Cell proliferation was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cell apoptosis was assessed with flow cytometry. The activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), endothelial nitric oxide synthase (eNOS), induced nitric oxide synthase (iNOS), and concentrations of malondialdehyde (MDA), glutathione (GSH), and protein carbonyl in culture medium were determined with colorimetric method. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was detected by enzyme linked immunosorbent assay. The results showed that excess iodine induced abnormal morphologic changes of cells, inhibited cell proliferation, and increased apoptosis rate. Iodine also reduced the activity of SOD, GSH-Px, and concentrations of GSH and increased the concentrations of MDA and protein carbonyl in a dose-dependent manner. Moreover, excess iodine decreased the activity of eNOS and increased the activity of iNOS and the expression of ICAM-1 and VCAM-1 in culture medium. Our results suggested that excess iodine exposure increased oxidative stress, caused damage of vascular endothelial cells, and altered the expression of adhesion factors and the activity of NOS. These changes may explain the mechanisms underlying excess iodine-induced vascular injury.

  13. Brown spider venom toxins interact with cell surface and are endocytosed by rabbit endothelial cells.

    Science.gov (United States)

    Nowatzki, Jenifer; de Sene, Reginaldo Vieira; Paludo, Katia Sabrina; Veiga, Silvio Sanches; Oliver, Constance; Jamur, Maria Célia; Nader, Helena Bonciani; Trindade, Edvaldo S; Franco, Célia Regina C

    2010-09-15

    Bites from the Loxosceles genus (brown spiders) cause severe clinical symptoms, including dermonecrotic injury, hemorrhage, hemolysis, platelet aggregation and renal failure. Histological findings of dermonecrotic lesions in animals exposed to Loxosceles intermedia venom show numerous vascular alterations. Study of the hemorrhagic consequences of the venom in endothelial cells has demonstrated that the degeneration of blood vessels results not only from degradation of the extracellular matrix molecule or massive leukocyte infiltration, but also from a direct and primary activity of the venom on endothelial cells. Exposure of an endothelial cell line in vitro to L. intermedia venom induce morphological alterations, such as cell retraction and disadhesion to the extracellular matrix. The aim of the present study was to investigate the interaction between the venom toxins and the endothelial cell surface and their possible internalization, in order to illuminate the information about the deleterious effect triggered by venom. After treating endothelial cells with venom toxins, we observed that the venom interacts with cell surface. Venom treatment also can cause a reduction of cell surface glycoconjugates. When cells were permeabilized, it was possible to verify that some venom toxins were internalized by the endothelial cells. The venom internalization involves endocytic vesicles and the venom was detected in the lysosomes. However, no damage to lysosomal integrity was observed, suggesting that the cytotoxic effect evoked by L. intermedia venom on endothelial cells is not mediated by venom internalization.

  14. The effects of TSH on human vascular endothelial cells and smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    田利民

    2014-01-01

    Objective To study the effect of thyroid-stimulating hormone(TSH)on human vascular endothelial cells and smooth muscle cells and to explore the roles of TSH in the development of atherosclerosis.Methods Human vascular endothelial cells and smooth muscle cells were cultured in vitro.MTT method was used to assay the effect of TSH on cell viability.Real-time PCR was used

  15. A reproducible automated segmentation algorithm for corneal epithelium cell images from in vivo laser scanning confocal microscopy.

    Science.gov (United States)

    Bullet, Julien; Gaujoux, Thomas; Borderie, Vincent; Bloch, Isabelle; Laroche, Laurent

    2014-06-01

    To evaluate an automated process to find borders of corneal basal epithelial cells in pictures obtained from in vivo laser scanning confocal microscopy (Heidelberg Retina Tomograph III with Rostock corneal module). On a sample of 20 normal corneal epithelial pictures, images were segmented through an automated four-step segmentation algorithm. Steps of the algorithm included noise reduction through a fast Fourier transform (FFT) band-pass filter, image binarization with a mean value threshold, watershed segmentation algorithm on distance map to separate fused cells and Voronoi diagram segmentation algorithm (which gives a final mask of cell borders). Cells were then automatically counted using this border mask. On the original image either with contrast enhancement or noise reduction, cells were manually counted by a trained operator. The average cell density was 7722.5 cells/mm(2) as assessed by automated analysis and 7732.5 cells/mm(2) as assessed by manual analysis (p = 0.93). Correlation between automated and manual analysis was strong (r = 0.974 [0.934-0.990], p mean difference in density of 10 cells/mm(2) and a limits of agreement ranging from -971 to +991 cells/mm(2) . Visually, the algorithm correctly found almost all borders. This automated segmentation algorithm is worth for assessing corneal epithelial basal cell density and morphometry. This procedure is fully reproducible, with no operator-induced variability. © 2014 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  16. Characterization of Bioeffects on Endothelial Cells under Acoustic Droplet Vaporization.

    Science.gov (United States)

    Seda, Robinson; Li, David S; Fowlkes, J Brian; Bull, Joseph L

    2015-12-01

    Gas embolotherapy is achieved by locally vaporizing microdroplets through acoustic droplet vaporization, which results in bubbles that are large enough to occlude blood flow directed to tumors. Endothelial cells, lining blood vessels, can be affected by these vaporization events, resulting in cell injury and cell death. An idealized monolayer of endothelial cells was subjected to acoustic droplet vaporization using a 3.5-MHz transducer and dodecafluoropentane droplets. Treatments included insonation pressures that varied from 2 to 8 MPa (rarefactional) and pulse lengths that varied from 4 to 16 input cycles. The bubble cloud generated was directly dependent on pressure, but not on pulse length. Cellular damage increased with increasing bubble cloud size, but was limited to the bubble cloud area. These results suggest that vaporization near the endothelium may impact the vessel wall, an effect that could be either deleterious or beneficial depending on the intended overall therapeutic application.

  17. ANTIBODIES DEFINING RAT ENDOTHELIAL-CELLS - RECA-1, A PAN-ENDOTHELIAL CELL-SPECIFIC MONOCLONAL-ANTIBODY

    NARCIS (Netherlands)

    DUIJVESTIJN, AM; VANGOOR, H; KLATTER, F; MAJOOR, GD; VANBUSSEL, E; VRIESMAN, PJCV

    1992-01-01

    We have been searching for antibodies reactive with rat endothelial cells. Two monoclonal antibodies (mAb), named RECA-1 and RECA-2 were produced and tested in immunoperoxidase staining on frozen sections of various rat tissues. Staining patterns were compared to those obtained with the mAbs OX-2, O

  18. The chemotactic activity of beta-carotene in endothelial cell progenitors and human umbilical vein endothelial cells: A microarray analysis

    NARCIS (Netherlands)

    Polus, A.; Kiec-wilk, B.; Hartwich, J.; Balwierz, A.; Stachura, J.; Dyduch, G.; Laidler, P.; Zagajewski, J.; Langman, T.; Schmitz, G.; Goralcsky, R.; Wertz, K.; Riss, G.; Keijer, J.; Dembinska-Kiec, A.

    2006-01-01

    Objectives: Endothelial cells and their progenitors play an important role in angiogenesis that is essential for organogenesis and tissue remodelling, as well as for inflammatory responses and carcinogenesis in all periods of life. In the present study, the authors concentrated on the direct effect

  19. ANTIBODIES DEFINING RAT ENDOTHELIAL-CELLS - RECA-1, A PAN-ENDOTHELIAL CELL-SPECIFIC MONOCLONAL-ANTIBODY

    NARCIS (Netherlands)

    DUIJVESTIJN, AM; VANGOOR, H; KLATTER, F; MAJOOR, GD; VANBUSSEL, E; VRIESMAN, PJCV

    1992-01-01

    We have been searching for antibodies reactive with rat endothelial cells. Two monoclonal antibodies (mAb), named RECA-1 and RECA-2 were produced and tested in immunoperoxidase staining on frozen sections of various rat tissues. Staining patterns were compared to those obtained with the mAbs OX-2, O

  20. Expression of basic fibroblast growth factor in rabbit corneal alkali wounds in the presence and absence of granulocytes.

    Science.gov (United States)

    Gan, Lisha; Fagerholm, Per; Palmblad, Jan

    2005-06-01

    To study the expression of basic fibroblast growth factor (bFGF) in the early phases of corneal wound healing in the presence or absence of granulocytes. A central penetrating corneal alkali wound was inflicted to one eye in each of 14 rabbits under general anaesthesia. Subsequently, seven of the rabbits were given fucoidin i.v. for 36 hours in order to block the selectins on the vascular endothelium, thus preventing blood granulocytes from entering the tissues. Then, corneas were prepared, stained for bFGF and evaluated by light microscopy. Whereas normal corneal epithelium expressed bFGF weakly, conjunctival epithelium did so strongly, particularly the goblet cells. The corneal endothelium showed medium staining, while keratocytes and vascular endothelial cells did not consistently express bFGF. After 36 hours of wound healing, a marked up-regulation of bFGF expression was observed in the corneal epithelial and endothelial cells, as well as in the keratocytes, that were migrating into the wound. No other changes were noted. None of these features were modulated when granulocyte emigration was prevented by fucoidin administration. The difference in bFGF expression between the corneal and conjunctival epithelium suggests a role for this growth factor in the barrier function at the limbus. Moreover, the specific presence of bFGF in cells migrating into the wound indicates the participation of bFGF in corneal wound healing. Expression of bFGF was independent of granulocytes.

  1. Morphology of primary human venous endothelial cell cultures before and after culture medium exchange.

    Science.gov (United States)

    Krüger-Genge, A; Fuhrmann, R; Jung, F; Franke, R P

    2015-01-01

    The evaluation of the interaction of human, venous endothelial cells (HUVEC) with body foreign materials on the cellular level cannot be performed in vivo, but is investigated in vitro under standard culture conditions. To maintain the vitality, proliferation and morphology of HUVEC seeded on body foreign substrates over days, the cell culture medium is usually exchanged every second day. It is well known, that alterations in the microenvironment of cells bear the risk of influencing cell morphology and function. In the current study the influence of cell culture medium exchange on HUVEC cytoskeletal microfilament structure and function was investigated. HUVEC in the third passage were seeded on extracellular matrix (ECM) - which was secreted from bovine corneal endothelial cells on glass- until functional confluence was reached. The experiment started 11 days after HUVEC seeding with an exchange of the cell culture medium followed by a staining of the actin microfilaments with phalloidin-rhodamin 1.5 and 5 minutes after medium exchange. The microfilaments were documented by use of an Olympus microscope (IMT-2) equipped with a UV lamp and online connected to a TV chain (Sony XC 50 ST/monochrome) implying an OPTIMAS - Image analysis system. Prostacyclin was analysed in the cell culture supernatant. 1.5 min after culture medium exchange in the functionally confluent cultures a slight disturbance of the actin microfilament structure with a broadening of the marginal filament band, a partial disconnection of cell-cell contacts and the appearance of intercellular fenestrations were observed. 5 minutes after medium exchange a redevelopment of the slightly disturbed microfilament structure with a condensation and narrowing of the marginal filament band was seen. 12 h later a further consolidation of the microfilament structure occurred. In addition, a perturbation of the cultured HUVEC occurred after cell culture medium exchange. The prostacyclin concentration in the

  2. Nylon-3 polymers that enable selective culture of endothelial cells.

    Science.gov (United States)

    Liu, Runhui; Chen, Xinyu; Gellman, Samuel H; Masters, Kristyn S

    2013-11-06

    Substrates that selectively encourage the growth of specific cell types are valuable for the engineering of complex tissues. Some cell-selective peptides have been identified from extracellular matrix proteins; these peptides have proven useful for biomaterials-based approaches to tissue repair or regeneration. However, there are very few examples of synthetic materials that display selectivity in supporting cell growth. We describe nylon-3 polymers that support in vitro culture of endothelial cells but do not support the culture of smooth muscle cells or fibroblasts. These materials may be promising for vascular biomaterials applications.

  3. An Important Method in the Investigation of Vascular Pathologies: Endothelial Cell Culture

    Directory of Open Access Journals (Sweden)

    Yusufhan Yazır

    2012-12-01

    Full Text Available Endothelial cells line the interior surface of blood vessels and form an interface between circulating blood in the lumen and the rest of the vessel wall. Endothelial cells are involved in many aspects of vascular biology, including barrier function, vasoconstriction, coagulation and inflamation. The endothelial cells in different organs have different functions and surface phenotype. These cells express prostoglandin-I2, platelet activating factor, collagen, endothelin-1, laminin, fibronectin and growth factors including platelet derived growth factor, fibroblast growth factor. İn the cell culture, cells can be isolated, maintened and proliferate in the laboratory conditions. The techniques of the cell culture have allowed scientists to use the cells in vitro for experimental studies, such as the production of vaccine, antibody and enzime, drug research, cell-cell interactions. Human umbilical vein endothelial cell is a good source for endothelial cell, because it is cheaper, easy to find and has the basic features of the normal endothelial cells.

  4. Arterial identity of endothelial cells is controlled by local cues.

    Science.gov (United States)

    Othman-Hassan, K; Patel, K; Papoutsi, M; Rodriguez-Niedenführ, M; Christ, B; Wilting, J

    2001-09-15

    The ephrins and their Eph receptors comprise the largest family of receptor tyrosine kinases. Studies on mice have revealed an important function of ephrin-B2 and Eph-B4 for the development of the arterial and venous vasculature, respectively, but the mechanisms regulating their expression have not been studied yet. We have cloned a chick ephrin-B2 cDNA probe. Expression was observed in endothelial cells of extra- and intraembryonic arteries and arterioles in all embryos studied from day 2 (stage 10 HH, before perfusion of the vessels) to day 16. Additionally, expression was found in the somites and neural tube in early stages, and later also in the smooth muscle cells of the aorta, parts of the Müllerian duct, dosal neural tube, and joints of the limbs. We isolated endothelial cells from the internal carotid artery and the vena cava of 14-day-old quail embryos and grafted them separately into day-3 chick embryos. Reincubation was performed until day 6 and the quail endothelial cells were identified with the QH1 antibody. The grafted arterial and venous endothelial cells expressed ephrin-B2 when they integrated into the lining of arteries. Cells that were not integrated into vessels, or into vessels other than arteries, were ephrin-B2-negative. The studies show that the expression of the arterial marker ephrin-B2 is controlled by local cues in arterial vessels of older embryos. Physical forces or the media smooth muscle cells may be involved in this process.

  5. Propofol protects against high glucose-induced endothelial adhesion molecules expression in human umbilical vein endothelial cells

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    Zhu Minmin

    2013-01-01

    Full Text Available Abstract Background Hyperglycemia could induce oxidative stress, activate transcription factor nuclear factor kappa B (NF-κB, up-regulate expression of endothelial adhesion molecules, and lead to endothelial injury. Studies have indicated that propofol could attenuate oxidative stress and suppress NF-κB activation in some situations. In the present study, we examined whether and how propofol improved high glucose-induced up-regulation of endothelial adhesion molecules in human umbilical vein endothelial cells (HUVECs. Methods Protein expression of endothelial adhesion molecules, NF-κB, inhibitory subunit of NF-κBα (IκBα, protein kinase Cβ2 (PKCβ2, and phosphorylation of PKCβ2 (Ser660 were measured by Western blot. NF-κB activity was measured by electrophoretic mobility shift assay. PKC activity was measured with SignaTECT PKC assay system. Superoxide anion (O2.- accumulation was measured with the reduction of ferricytochrome c assay. Human peripheral mononuclear cells were prepared with Histopaque-1077 solution. Results High glucose induced the expression of endothelial selectin (E-selectin, intercellular adhesion molecule 1 (ICAM-1, vascular cell adhesion molecule 1 (VCAM-1, and increased mononuclear-endothelial adhesion. High glucose induced O2.- accumulation, PKCβ2 phosphorylation and PKC activation. Further, high glucose decreased IκBα expression in cytoplasm, increased the translocation of NF-κB from cytoplasm to nuclear, and induced NF-κB activation. Importantly, we found these high glucose-mediated effects were attenuated by propofol pretreatment. Moreover, CGP53353, a selective PKCβ2 inhibitor, decreased high glucose-induced NF-κB activation, adhesion molecules expression, and mononuclear-endothelial adhesion. Conclusion Propofol, via decreasing O2.- accumulation, down-regulating PKCβ2 Ser660 phosphorylation and PKC as well as NF-κB activity, attenuated high glucose-induced endothelial adhesion molecules expression

  6. Superficial keratectomy and topical mitomycin C as therapy for a corneal squamous cell carcinoma in a dog.

    Science.gov (United States)

    Karasawa, K; Matsuda, H; Tanaka, A

    2008-04-01

    A 10-year-old female West Highland white terrier was presented with refractory hyperplastic keratitis of the left cornea of one month's duration. At this time, a vascularised and rough lesion 5 mm in diameter was observed on the left cornea. No other abnormality was recognised on the affected eye. The corneal neoplasm was surgically removed and histologically diagnosed as a squamous cell carcinoma. For two months after the surgery, 0.04 percent mitomycin C (MMC) eye drops were applied as adjuvant chemotherapy. Primary corneal squamous cell carcinoma with no history of keratoconjunctivitis sicca is rare in dogs. In the present report, surgical removal of the neoplasm was combined with the topical administration of the anticancer drug mitomycin C and a good prognosis was obtained. The result indicates that the combination treatment used in this case may be an appropriate therapeutic choice for corneal squamous cell carcinoma in dogs.

  7. Signal transduction pathways in mast cell granule-mediated endothelial cell activation

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    Luqi Chi

    2003-01-01

    Full Text Available Background: We have previously shown that incubation of human endothelial cells with mast cell granules results in potentiation of lipopolysaccharide-induced production of interleukin-6 and interleukin-8.

  8. System for tracking transplanted limbal epithelial stem cells in the treatment of corneal stem cell deficiency (Conference Presentation)

    Science.gov (United States)

    Boadi, Joseph; Matcher, Stephen; MacNeil, Sheila; Sangwan, Virender S.

    2016-04-01

    The prevailing hypothesis for the existence and healing of the avascular corneal epithelium is that this layer of cells are continually produced by stem cells in the limbus and transported onto the cornea to mature into corneal epithelium. In the event that the cornea is damaged and the limbal stem cell population is severely reduced, this condition known as Limbal Stem Cell Deficiency and can lead to blindness. There are numerous treatments but most have high long term failure rates. Most treatment methods include the transplantation of limbal stem cells into damaged limbus with hope of repopulating the region and regenerating at healthy corneal epithelium. Optical Coherence Tomography (OCT) is well known for its high resolution in vivo images. A bespoke OCT has been built to investigate the trajectories of these limbal stem cells after transplantation to see whether if they do repopulate the damaged limbus or not. In the experimentation magneto-labelling was used to track the limbal stem cells. For the magneto-labelling a mixture of limbal stem cells and cornea epithelium are cultured with super paramagnetic iron (Fe3O4) nanoparticles (20-30nm in size) for 24hours, to allow for uptake. The cells are then transplanted onto the denuded cornea. The transplanted cell mixture with the encapsulated magnetic nanoparticles is actuated with an external magnetic field 0.08T leading to a phase modulation on the signal. A Phase sensitive Magneto-motive OCT is used to locate the transplanted cells. The location of the cells with embed SPIOs were located both in 2D and 3D.

  9. Isolation of Endothelial Cells and Vascular Smooth Muscle Cells from Internal Mammary Artery Tissue

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    Moss, Stephanie C.; Bates, Michael; Parrino, Patrick E.; Woods, T. Cooper

    2007-01-01

    Analyses of vascular smooth muscle cell and endothelial cell function through tissue culture techniques are often employed to investigate the underlying mechanisms regulating cardiovascular disease. As diseases such as diabetes mellitus and chronic kidney disease increase a patient's risk of cardiovascular disease, the development of methods for examining the effects of these diseases on vascular smooth muscle cells and endothelial cells is needed. Commercial sources of endothelial cells and vascular smooth muscle cells generally provide minimal donor information and are in limited supply. This study was designed to determine if vascular smooth muscle cells and endothelial cells could be isolated from human internal mammary arteries obtained from donors undergoing coronary artery bypass graft surgery. As coronary artery bypass graft surgery is a commonly performed procedure, this method would provide a new source for these cells that when combined with the donor's medical history will greatly enhance our studies of the effects of complicating diseases on vascular biology. Internal mammary artery tissue was obtained from patients undergoing coronary artery bypass graft surgery. Through a simple method employing two separate tissue digestions, vascular smooth muscle cells and endothelial cells were isolated and characterized. The isolated vascular smooth muscle cells and endothelial cells exhibited the expected morphology and were able to be passaged for further analysis. The vascular smooth muscle cells exhibited positive staining for α-smooth muscle actin and the endothelial cells exhibited positive staining for CD31. The overall purity of the isolations was > 95%. This method allows for the isolation of endothelial cells and vascular smooth muscle cells from internal mammary arteries, providing a new tool for investigations into the interplay of vascular diseases and complicating diseases such as diabetes and kidney disease. PMID:21603530

  10. Nitrones reverse hyperglycemia-induced endothelial dysfunction in bovine aortic endothelial cells.

    Science.gov (United States)

    Headley, Colwyn A; DiSilvestro, David; Bryant, Kelsey E; Hemann, Craig; Chen, Chun-An; Das, Amlan; Ziouzenkova, Ouliana; Durand, Grégory; Villamena, Frederick A

    2016-03-15

    Hyperglycemia has been implicated in the development of endothelial dysfunction through heightened ROS production. Since nitrones reverse endothelial nitric oxide synthase (eNOS) dysfunction, increase antioxidant enzyme activity, and suppress pro-apoptotic signaling pathway and mitochondrial dysfunction from ROS-induced toxicity, the objective of this study was to determine whether nitrone spin traps DMPO, PBN and PBN-LA were effective at duplicating these effects and improving glucose uptake in an in vitro model of hyperglycemia-induced dysfunction using bovine aortic endothelial cells (BAEC). BAEC were cultured in DMEM medium with low (5.5mM glucose, LG) or high glucose (50mM, HG) for 14 days to model in vivo hyperglycemia as experienced in humans with metabolic disease. Improvements in cell viability, intracellular oxidative stress, NO and tetrahydrobiopterin (BH4)​ levels, mitochondrial membrane potential, glucose transport, and activity of antioxidant enzymes were measured from single treatment of BAEC with nitrones for 24h after hyperglycemia. Chronic hyperglycemia significantly increased intracellular ROS by 50%, decreased cell viability by 25%, reduced NO bioavailability by 50%, and decreased (BH4) levels by 15% thereby decreasing NO production. Intracellular glucose transport and superoxide dismutase (SOD) activity were also decreased by 50% and 25% respectively. Nitrone (PBN and DMPO, 50 μM) treatment of BAEC grown in hyperglycemic conditions resulted in the normalization of outcome measures except for SOD and catalase activities. Our findings demonstrate that the nitrones reverse the deleterious effects of hyperglycemia in BAEC. We believe that in vivo testing of these nitrone compounds in models of cardiometabolic disease is warranted.

  11. Coculture of dorsal root ganglion neurons and differentiated human corneal stromal stem cells on silk-based scaffolds.

    Science.gov (United States)

    Wang, Siran; Ghezzi, Chiara E; White, James D; Kaplan, David L

    2015-10-01

    Corneal tissue displays the highest peripheral nerve density in the human body. Engineering of biomaterials to promote interactions between neurons and corneal tissue could provide tissue models for nerve/cornea development, platforms for drug screening, as well as innovative opportunities to regenerate cornea tissue. The focus of this study was to develop a coculture system for differentiated human corneal stromal stem cells (dhCSSCs) and dorsal root ganglion neurons (DRG) to mimic the human cornea tissue interactions. Axon extension, connectivity, and neuron cell viability were studied. DRG neurons developed longer axons when cocultured with dhCSSCs in comparison to neuron cultures alone. To assess the mechanism involved in the coculture response, nerve growth factors (NGF) secreted by dhCSSCs including NGF, brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), and neurotrophin-3 were characterized with greater focus on BDNF secretion. DhCSSCs also secreted collagen type I, an extracellular matrix molecule favorable for neuronal outgrowth. This coculture system provides a slowly degrading silk matrix to study neuronal responses in concert with hCSSCs related to innervation of corneal tissue with utility toward human corneal nerve regeneration and associated diseases. © 2015 Wiley Periodicals, Inc.

  12. Antiproliferative Effects of Drugs on Endothelial and Osteoblastic Cells and Altered Release of Angioregulatory Mediators by Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Hilde Kvestad

    2014-01-01

    Full Text Available The combined use of the histone deacetylase inhibitor valproic acid (VPA, the retinoic acid receptor-α agonist all-trans retinoic acid (ATRA, and the deoxyribonucleic acid polymerase-α inhibitor cytarabine (Ara-C is now considered for disease-stabilizing treatment of acute myeloid leukemia (AML. Leukemogenesis and leukemia cell chemoresistance seem to be supported by neighbouring stromal cells in the bone marrow, and we have therefore investigated the effects of these drugs on primary human endothelial cells and the osteoblastic Cal72 cell line. The results show that VPA and Ara-C have antiproliferative effects, and the antiproliferative/cytotoxic effect of Ara-C was seen at low concentrations corresponding to serum levels found during low-dose in vivo treatment. Furthermore, in functional assays of endothelial migration and tube formation VPA elicited an antiangiogenic effect, whereas ATRA elicited a proangiogenic effect. Finally, VPA and ATRA altered the endothelial cell release of angiogenic mediators; ATRA increased levels of CXCL8, PDGF-AA, and VEGF-D, while VPA decreased VEGF-D and PDGF-AA/BB levels and both drugs reduced MMP-2 levels. Several of these mediators can enhance AML cell proliferation and/or are involved in AML-induced bone marrow angiogenesis, and direct pharmacological effects on stromal cells may thus indirectly contribute to the overall antileukemic activity of this triple drug combination.

  13. Suprabasin as a novel tumor endothelial cell marker

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    Alam, Mohammad T; Nagao-Kitamoto, Hiroko; Ohga, Noritaka; Akiyama, Kosuke; Maishi, Nako; Kawamoto, Taisuke; Shinohara, Nobuo; Taketomi, Akinobu; Shindoh, Masanobu; Hida, Yasuhiro; Hida, Kyoko

    2014-01-01

    Recent studies have reported that stromal cells contribute to tumor progression. We previously demonstrated that tumor endothelial cells (TEC) characteristics were different from those of normal endothelial cells (NEC). Furthermore, we performed gene profile analysis in TEC and NEC, revealing that suprabasin (SBSN) was upregulated in TEC compared with NEC. However, its role in TEC is still unknown. Here we showed that SBSN expression was higher in isolated human and mouse TEC than in NEC. SBSN knockdown inhibited the migration and tube formation ability of TEC. We also showed that the AKT pathway was a downstream factor of SBSN. These findings suggest that SBSN is involved in the angiogenic potential of TEC and may be a novel TEC marker. PMID:25283635

  14. Rho/ROCK signaling in regulation of corneal epithelial cell cycle progression.

    Science.gov (United States)

    Chen, Jian; Guerriero, Emily; Lathrop, Kira; SundarRaj, Nirmala

    2008-01-01

    The authors' previous study showed that the expression of a Rho-associated serine/threonine kinase (ROCK) is regulated during cell cycle progression in corneal epithelial cells. The present study was conducted to determine whether and how Rho/ROCK signaling regulates cell cycle progression. Rabbit corneal epithelial cells (RCECs) in culture were arrested in the G(0) phase of the cell cycle by serum deprivation and then allowed to re-enter the cell cycle in the presence or absence of the ROCK inhibitor (Y27632) in serum-supplemented medium. The number of cells in the S phase, the relative levels of specific cyclins and CDKs and their intracellular distribution, and the relative levels of mRNAs were determined by BrdU labeling, Western blot and immunocytochemical analyses, and real-time RT-PCR, respectively. ROCK inhibition delayed the progression of G(1) to S phase and led to a decrease in the number of RCECs entering the S phase between 12 and 24 hours from 31.5% +/- 4.5% to 8.1% +/- 2.6%. During the cell cycle progression, protein and mRNA levels of cyclin-D1 and -D3 and cyclin-dependent kinases CDK4 and CDK6 were significantly lower, whereas the protein levels of the CDK inhibitor p27(Kip1) were higher in ROCK-inhibited cells. Intracellular mRNA or protein levels of cyclin-E and protein levels of CDK2 were not significantly affected, but their nuclear translocation was delayed by ROCK inhibition. ROCK signaling is involved in cell cycle progression in RCECs, possibly by upregulation of cyclin-D1 and -D3 and CDK4, -6, and -2; nuclear translocation of CDK2 and cyclin-E; and downregulation of p27(Kip1).

  15. Leptin-induced transphosphorylation of vascular endothelial growth factor receptor increases Notch and stimulates endothelial cell angiogenic transformation.

    Science.gov (United States)

    Lanier, Viola; Gillespie, Corey; Leffers, Merle; Daley-Brown, Danielle; Milner, Joy; Lipsey, Crystal; Webb, Nia; Anderson, Leonard M; Newman, Gale; Waltenberger, Johannes; Gonzalez-Perez, Ruben Rene

    2016-10-01

    Leptin increases vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), and Notch expression in cancer cells, and transphosphorylates VEGFR-2 in endothelial cells. However, the mechanisms involved in leptin's actions in endothelial cells are not completely known. Here we investigated whether a leptin-VEGFR-Notch axis is involved in these leptin's actions. To this end, human umbilical vein and porcine aortic endothelial cells (wild type and genetically modified to overexpress VEGFR-1 or -2) were cultured in the absence of VEGF and treated with leptin and inhibitors of Notch (gamma-secretase inhibitors: DAPT and S2188, and silencing RNA), VEGFR (kinase inhibitor: SU5416, and silencing RNA) and leptin receptor, OB-R (pegylated leptin peptide receptor antagonist 2: PEG-LPrA2). Interestingly, in the absence of VEGF, leptin induced the expression of several components of Notch signaling pathway in endothelial cells. Inhibition of VEGFR and Notch signaling significantly decreased leptin-induced S-phase progression, proliferation, and tube formation in endothelial cells. Moreover, leptin/OB-R induced transphosphorylation of VEGFR-1 and VEGFR-2 was essential for leptin's effects. These results unveil for the first time a novel mechanism by which leptin could induce angiogenic features via upregulation/trans-activation of VEGFR and downstream expression/activation of Notch in endothelial cells. Thus, high levels of leptin found in overweight and obese patients might lead to increased angiogenesis by activating VEGFR-Notch signaling crosstalk in endothelial cells. These observations might be highly relevant for obese patients with cancer, where leptin/VEGFR/Notch crosstalk could play an important role in cancer growth, and could be a new target for the control of tumor angiogenesis.

  16. Brain microvascular endothelial cell transplantation ameliorates ischemic white matter damage.

    Science.gov (United States)

    Puentes, Sandra; Kurachi, Masashi; Shibasaki, Koji; Naruse, Masae; Yoshimoto, Yuhei; Mikuni, Masahiko; Imai, Hideaki; Ishizaki, Yasuki

    2012-08-21

    Ischemic insults affecting the internal capsule result in sensory-motor disabilities which adversely affect the patient's life. Cerebral endothelial cells have been reported to exert a protective effect against brain damage, so the transplantation of healthy endothelial cells might have a beneficial effect on the outcome of ischemic brain damage. In this study, endothelin-1 (ET-1) was injected into the rat internal capsule to induce lacunar infarction. Seven days after ET-1 injection, microvascular endothelial cells (MVECs) were transplanted into the internal capsule. Meningeal cells or 0.2% bovine serum albumin-Hank's balanced salt solution were injected as controls. Two weeks later, the footprint test and histochemical analysis were performed. We found that MVEC transplantation improved the behavioral outcome based on recovery of hind-limb rotation angle (P<0.01) and induced remyelination (P<0.01) compared with the control groups. Also the inflammatory response was repressed by MVEC transplantation, judging from fewer ED-1-positive activated microglial cells in the MVEC-transplanted group than in the other groups. Elucidation of the mechanisms by which MVECs ameliorate ischemic damage of the white matter may provide important information for the development of effective therapies for white matter ischemia.

  17. Coniferyl Aldehyde Ameliorates Radiation Intestine Injury via Endothelial Cell Survival

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    Jeong, Ye Ji; Jung, Myung Gu; Lee, Yoonjin; Lee, Haejune [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Lee, Yunsil [Ewha Woman' s Univ., Seoul (Korea, Republic of); Ko, Younggyu [Korea Univ., Seoul (Korea, Republic of)

    2014-05-15

    Cancer treatments related gastrointestinal toxicity has also been recognized as a significant economic burden. Especially, extensive apoptosis of microvascular endothelial cell of the lamina propria is the primary lesion initiating intestinal radiation damage after abdominal radiation therapy. Coniferyl aldehyde (CA) is phenolic compounds isolated from cork stoppers, and one of the major pyrolysis products of lignin. Shi H. was support for the empirical use of CA as a medicinal food for cardiovascular diseases. CA has positive effect in broad way but there is no consequence in radiation induced intestine damage. Here, we investigate effect of CA on small intestine after abdominal IR to mice in this study. In this study, CA increased the survival rate in C3H mice against 13.5 Gy abdominal IR. We found CA protects small intestine via preventing endothelial cell apoptosis and enhancing their angiogenic activity. CA also showed protective effect on crypt cell survival. Endothelial cell survival may affect crypt cell protection against IR. From this data, we concluded that CA is effective for protection against abdominal radiation injury. CA could ameliorate side-effect of radiation therapy.

  18. Protection of human corneal epithelial cells from TNF-α-induced disruption of barrier function by rebamipide.

    Science.gov (United States)

    Kimura, Kazuhiro; Morita, Yukiko; Orita, Tomoko; Haruta, Junpei; Takeji, Yasuhiro; Sonoda, Koh-Hei

    2013-04-17

    TNF-α disrupts the barrier function of cultured human corneal epithelial (HCE) cells. We investigated the effects of the cytoprotective drug rebamipide on this barrier disruption by TNF-α as well as on corneal epithelial damage in a rat model of dry eye. The barrier function of HCE cells was evaluated by measurement of transepithelial electrical resistance. The distribution of tight-junction (ZO-1, occludin) and adherens-junction (E-cadherin, β-catenin) proteins, and the p65 subunit of nuclear factor-κB (NF-κB) was determined by immunofluorescence microscopy. Expression of junctional proteins as well as phosphorylation of the NF-κB inhibitor IκB-α and myosin light chain (MLC) were examined by immunoblot analysis. A rat model of dry eye was developed by surgical removal of exorbital lacrimal glands. Rebamipide inhibited the disruption of barrier function as well as the downregulation of ZO-1 expression, and the disappearance of ZO-1 from the interfaces of neighboring HCE cells induced by TNF-α. It also inhibited the phosphorylation and downregulation of IκB-α, the translocation of p65 to the nucleus, the formation of actin stress fibers, and the phosphorylation of MLC induced by TNF-α in HCE cells. Treatment with rebamipide eyedrops promoted the healing of corneal epithelial defects as well as attenuated the loss of ZO-1 from the surface of corneal epithelial cells in rats. Rebamipide protects corneal epithelial cells from the TNF-α-induced disruption of barrier function by maintaining the distribution and expression of ZO-1 as well as the organization of the actin cytoskeleton. Rebamipide is, thus, a potential drug for preventing or ameliorating the loss of corneal epithelial barrier function associated with ocular inflammation.

  19. Pharmacologically active microcarriers for endothelial progenitor cell support and survival.

    Science.gov (United States)

    Musilli, Claudia; Karam, Jean-Pierre; Paccosi, Sara; Muscari, Claudio; Mugelli, Alessandro; Montero-Menei, Claudia N; Parenti, Astrid

    2012-08-01

    The regenerative potential of endothelial progenitor cell (EPC)-based therapies is limited due to poor cell viability and minimal retention following application. Neovascularization can be improved by means of scaffolds supporting EPCs. The aim of the present study was to investigate whether human early EPCs (eEPCs) could be efficiently cultured on pharmacologically active microcarriers (PAMs), made with poly(d,l-lactic-coglycolic acid) and coated with adhesion/extracellular matrix molecules. They may serve as a support for stem cells and may be used as cell carriers providing a controlled delivery of active protein such as the angiogenic factor, vascular endothelial growth factor-A (VEGF-A). eEPC adhesion to fibronectin-coated PAMs (FN-PAMs) was assessed by means of microscopic evaluation and by means of Alamar blue assay. Phospho ERK(1/2) and PARP-1 expression was measured by means of Western blot to assess the survival effects of FN-PAMs releasing VEGF-A (FN-VEGF-PAMs). The Alamar blue assay or a modified Boyden chamber assay was employed to assess proliferative or migratory capacity, respectively. Our data indicate that eEPCs were able to adhere to empty FN-PAMs within a few hours. FN-VEGF-PAMs increased the ability of eEPCs to adhere to them and strongly supported endothelial-like phenotype and cell survival. Moreover, the release of VEGF-A by FN-PAMs stimulated in vitro HUVEC migration and proliferation. These data strongly support the use of PAMs for supporting eEPC growth and survival and for stimulating resident mature human endothelial cells.

  20. Limbal Stem Cell Transplantation for Gelatinous Drop-like Corneal Dystrophy

    Directory of Open Access Journals (Sweden)

    Hossein Movahedan

    2013-01-01

    Full Text Available Purpose: To report the outcomes of allograft limbal stem cell transplantation for recurrent gelatinous drop-like corneal dystrophy (GDLD. Methods: In this non-comparative interventional case series, 4 eyes of 3 consecutive patients with recurrent GDLD underwent allograft limbal stem cell transplantation. Two eyes underwent concomitant penetrating keratoplasty while the other two underwent simultaneous superficial keratectomy.