Biomedical subjects

Lung-Kun Yeh

Publications and source records attributed to Lung-Kun Yeh.

9 recordsLinked to original sources

PubMed2006

Pax6 overexpression suppresses cell proliferation and retards the cell cycle in corneal epithelial cells.

PURPOSE: The gene encoding transcription factor Pax6 resides at the top of a genetic hierarchy controlling development and morphogenesis of the eye. Pax6 continues to be expressed in the ocular surface epithelia of the postnatal eye. The goal of this study was to investigate a possible role for Pax6 in controlling dynamics of the ocular surface epithelia. METHODS: Full-length mouse Pax6 (mPax) cDNA, or truncated mPax6Delta286 lacking the transcriptional activation domain was inserted into a tetracycline-inducible vector (Tet-on). A rabbit corneal epithelial cell line SIRC was used to establish stable transformants. Induction of Pax6 or truncated Pax6Delta286 proteins by doxycycline (DOX) was examined by Western blot and immunohistochemistry. The effects of Pax6 overexpression on cell cycle progression were assessed by the cell proliferation index, cell growth curve, and cell cycle assay. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay was performed to detect apoptotic cells. Recombinant adenovirus-carrying mPax6 or mPax6Delta286 transgenes were used for transient transduction of primary rabbit corneal epithelial cells, and the effect on cell cycle progression was assayed. RESULTS: The level of Pax6 or truncated Pax6 was tightly regulated by DOX. Overexpression of full-length Pax6 retarded the rate of cell proliferation, whereas the truncated form had no effect. Full-length Pax6 affected the rate at which individual cells traversed the cell cycle and induced caspase-3-independent apoptosis in a small percentage of cells. Transient transduction of cells with recombinant mPax6 adenovirus also inhibited cell proliferation. CONCLUSIONS: Inhibition of cell proliferation in Pax6-overexpressing corneal epithelial cell lines and primary cell culture is consistent with the notion that Pax6 plays a role in controlling corneal epithelial cell dynamics in vivo.

Adenoviridae↗

PubMed2005

Keratocan expression of murine keratocytes is maintained on amniotic membrane by down-regulating transforming growth factor-beta signaling.

Keratocytes in the corneal stroma express keratan sulfate-containing proteoglycans including cornea-specific keratocan. On plastic dishes, human, bovine, and rabbit keratocytes lose their characteristic dendritic morphology and keratocan expression when cultured in serum-containing media. Herein, we demonstrated that murine keratocytes also acquired a fibroblastic shape and lost keratocan expression after first passage when cultured on plastic in the presence of serum. In contrast, cells expanded on human amniotic membrane (AM) stromal matrix maintained a three-dimensional dendritic morphology and expressed keratocan mRNA and protein for at least 8 passages before senescence. When keratocytes were cultured on AM, the promoter activity of transforming growth factor (TGF)-beta2 and TGF-beta receptor II was down-regulated as compared with that on plastic. Furthermore, cells on AM continuously retained Smad 2 and Smad 4 in the cytoplasm and did not express alpha-smooth muscle actin, even when 10 ng/ml TGF-beta1 was added in a serum-free medium for up to 5 days. In parallel to such down-regulation of TGF-beta signaling, keratocan promoter-driven ECFP expression was observed in cells cultured either on AM in the presence of serum or on plastic containing serum treated with a neutralizing antibody to TGF-beta. Collectively, these results indicate that down-regulation of Smad-mediated TGF-beta signaling is an important mechanism for cultured keratocytes to maintain a normal phenotype while continuously expanded in a serum-containing medium. This strategy of suppressing TGF-beta signaling, achieved by AM stromal matrix in part via suppression of TGF-beta gene transcription, can be used to expand keratocytes in culture without the use of AM in the future.

Animals↗

PubMed2005

Infectious keratitis related to overnight orthokeratology.

PURPOSE: To report the microbial culture results, clinical course, and visual outcomes for infectious keratitis related to overnight orthokeratology. METHODS: The records of patients with infectious keratitis related to overnight orthokeratology who presented to a tertiary referral center from April 2000 to March 2003 were retrospectively reviewed. RESULTS: Twenty patients (21 eyes) were included; 1 patient had bilateral infections. The average age of the patients was 14 years. The average period between the time the patient started the overnight orthokeratology program and the onset of infectious keratitis was 23 months. Thirteen of the 21 eyes were culture positive. Organisms cultured were Pseudomonas aeruginosa (n = 9), coagulase-negative Staphylococcus species (n = 2), Serratia marcescens (n = 1), and Acathamoeba species (n = 1). All patients responded well to medical antimicrobial treatment. Final best spectacle-corrected visual acuity ranged from 20/20 to 20/100. CONCLUSIONS: Infectious keratitis is a potential complication of overnight orthokeratology that may cause significant visual impairment. Parents of children who consider overnight orthokeratology should evaluate the benefit of temporary myopia reduction and the risk of infection.

Acanthamoeba↗

PubMed2005

Soluble lumican glycoprotein purified from human amniotic membrane promotes corneal epithelial wound healing.

PURPOSE: To purify and characterize the glycoprotein lumican, isolated from human amniotic membrane (AM), and to examine its efficacy in treating corneal epithelium debridement. METHODS: An affinity-purified, anti-human lumican antibody-conjugated protein A Sepharose column was used to purify soluble lumican protein from human AM. The purified AM lumican was characterized by two-dimensional and SDS gel electrophoresis, plus Western blot analysis with anti-lumican antibody. The effects of lumican on corneal epithelial wound healing were examined in an organ culture mouse eye model. RESULTS: Lumican was found to be abundantly present in the stroma of human AM. It was extracted from the AM by isotonic, 1 M NaCl, and 4 M guanidine HCl solutions, suggesting that it is present in both the soluble and matrix-bound states. In two-dimensional gel electrophoresis, the 50-kDa human amniotic lumican purified by antibody-conjugated affinity chromatography migrated in a smear between pH 3.0 and 6.0. After endo-beta-galactosidase digestion, it existed as a single core protein at pH 6.0, suggesting that native human amniotic lumican is a glycoprotein with short sugar moiety. Addition of purified human AM lumican to cultured medium promoted re-epithelialization and enhanced cell proliferation of wild-type mouse corneal epithelial cells in an organ culture. In lumican-knockout (lum(-/-)) mice, the effect of human lumican on promoting corneal epithelial wound healing was even more dramatic than in wild-type mice. CONCLUSIONS: The diversified functions of lumican include modulation of epithelial cells in wound healing and serving as an extracellular matrix component. Administration of lumican may be beneficial for treating epithelial defects in the cornea and other tissues.

Amnion↗

PubMed2005

The heterogeneous murine corneal stromal cell populations in vitro.

PURPOSE: To demonstrate that the murine corneal stroma is inhabited by heterogeneous cell populations that include cells expressing nestin. METHODS: Collagenase-isolated corneal stroma cells obtained from newborn and adult mice (2nd and 12th postnatal weeks, respectively), were seeded at low (5 cells/mm2), intermediate (50 cells/mm2), and high (500 cells/mm2) densities in DMEM/F12 containing insulin, transferrin, selenium, and 1% nonessential amino acids. Corneal stroma cells cultured at 500 cells/mm2 were treated with 10 ng/mL human recombinant transforming growth factor (TGF)-beta1 for 5 days. Cell morphology and expression of alpha-smooth muscle actin, choline acetyltransferase, CD45, glial fibrillary acidic protein (GFAP), keratocan, nestin, neurofilaments, protein gene product 9.5, tyrosine hydroxylase, and vimentin were examined. RESULTS: Phase-contrast microscopy demonstrated that freshly isolated corneal stromal cells are heterogeneous in morphology and include dendritic, stellate, neuronal, and small polyhedral cells. Immunostaining of primary cultures of 2- and 12-week-old mice, 24 hours after seeding at the intermediate density, showed that 100% of cells expressed vimentin and 97.7% +/- 2.7% expressed keratocan. alpha-Smooth muscle actin was expressed by 0.2% +/- 0.05% of cells in the 2-week-old group and 0.1% +/- 0.07% in 12-week-old group. Neurofilament was expressed by 0.5% +/- 0.03% and 0.7% +/- 0.03% of cells in the 2- and 12-week-old groups, respectively. No cell expressed GFAP or nestin. After 5 days in culture, cells seeded at high density aggregated as clusters that were immunoreactive to nestin in both groups. Cell clusters and migrating cells reacted to pgp 9.5, and migrating cells, but not the cell clusters, reacted to tyrosine hydroxylase. Cell cluster formation and nestin expression were abolished by culturing in the presence of TGF-beta1. CONCLUSIONS: Normal murine corneal stroma contains heterogeneous cell populations including cells with the potential to form clusters and express the progenitor marker nestin. This potential is disrupted by the addition of TGF-beta1 to the culture medium.

Actins↗

PubMed2004

How does amniotic membrane work?

Transplantation of amniotic membrane as a temporary or permanent graft promotes epithelial wound healing and exerts potent anti-inflammatory and anti-scarring effects on the ocular surface. These actions depend on the killing of allogeneic amniotic cells and preservation of the cytokine-containing matrix during the preparation of the amniotic membrane. This review describes how these actions inherently operate in utero and how amniotic membrane transplantation aims to recreate such a fetal environment to exert these actions by insulating the surgical site from the host environment. These actions also render the amniotic membrane a unique niche capable of expanding both epithelial and mesenchymal progenitor cells ex vivo, while maintaining their normal cell phenotypes. As a result, the amniotic membrane becomes an ideal substrate for engineering different types of ocular surface tissues for transplantation. Further studies investigating the exact molecular mechanism by which the amniotic membrane works will undoubtedly unravel additional applications in reconstruction and engineering of both ocular and nonocular tissues in the burgeoning field of regenerative medicine.

Journal Article↗

PubMed2004

Calcium-induced abnormal epidermal-like differentiation in cultures of mouse corneal-limbal epithelial cells.

PURPOSE: To develop a reproducible method for expanding mouse corneal-limbal epithelial cells and determine the role of extracellular Ca(2+) concentration and serum in modulating their growth and differentiation. METHODS: Intact and viable corneal epithelial sheets were isolated from CD-1 albino mouse eyeballs by incubating for 18 hours at 4 degrees C in 15 mg/mL dispase II with sorbitol in defined keratinocyte serum-free medium (KSFM) or supplementary hormonal epithelial medium (SHEM). These sheets were trypsinized into single cells and cultured on plastic in KSFM or SHEM. Cultures in KSFM were further manipulated by increasing Ca(2+) concentration to 0.9 mM, with or without 5% FBS. Epithelial growth was compared in KSFM/KSFM (digestion medium/culture medium) and SHEM/SHEM by continuous passaging at a 1:3 split and by crystal violet staining of confluent dishes. Epithelial differentiation was assessed by immunostaining and/or immunoblotting to ZO-1, cytokeratin K12 (K12), connexin 43 (Cx43), cytokeratin K10 (K10), and involucrin. RESULTS: Intact and viable corneal-limbal epithelial sheets were consistently isolated from more than 200 mouse eyes. Gradual increases in cell sizes and expression of ZO-1, K12, and Cx43 were noted from KSFM/KSFM to SHEM/KSFM, KSFM/SHEM, and SHEM/SHEM at passage 0. Epithelial growth ended at passage 1 in SHEM/SHEM but continued until passage 3 in KSFM/KSFM. Immunoblot analysis revealed that K12 expression was the highest in SHEM/SHEM, decreased from passages 0 to 1, and disappeared in passage 2 in KSFM/KSFM, with complete replacement of K10 and increasing expression of involucrin. Appearance of K10 was facilitated by 0.9 mM Ca(2+) but suppressed by 5% FBS in KSFM at passage 0. CONCLUSIONS: Mouse corneal-limbal epithelial sheets can be used for initiating primary cultures, and their differentiation is promoted, whereas growth is suppressed, by a high Ca(2+) concentration, even during enzymatic digestion. In serum-free medium, abnormal epidermal-like differentiation is promoted by increasing Ca(2+) concentrations but prevented by serum. These results provide the ability to devise a medium to promote growth while maintaining normal differentiation.

Animals↗

PubMed2004

Pseudomonas aeruginosa corneal ulcer related to overnight orthokeratology.

BACKGROUND: Overnight orthokeratology was thought to be a safe and non-invasive alternative for low-grade myopia and astigmatism correction. We assessed histories, clinical courses, and visual outcomes of the patients with pseudomonal keratitis related to overnight orthokeratology. METHODS: The records of six patients with pseudomonal keratitis related to overnight orthokeratology were reviewed from January 2001 through December 2002. RESULTS: The average age of the patients was 13 years. The average period between the time that the patient started the overnight orthokeratology program and the onset of infectious keratitis was 17 months. All patients presented with painful red eyes. The area of the corneal ulcer was central in three, and paracentral in three eyes. The corneal infiltrate was small in one eye, and medium in five eyes. The corneal scrapings from these six patients revealed Pseudomonas aeruginosa. All patients responded well to topical antibiotic treatment. Two of six eyes had a final visual acuity within two lines of the pre-infection vision at the last follow-up. Four of the eyes examined lost their best-corrected visual acuity due to central corneal scar or irregular astigmatism. CONCLUSIONS: Overnight orthokeratology contact lens wear has the potential complication of pseudomonal keratitis and may cause significant visual impairment.

Adolescent↗

PubMedDate not supplied

Sequels, complications and management of a chemical burn associated with cement splash.

We present a case of successful superficial keratectomy and amniotic membrane grafting to re-establish ocular surface from denuded stroma and significant limbal ischemia caused by a cement splash. We fully documented a case report about the sequels, complications and management strategies of a chemical burn to the eyes associated with a cement splash. Slit lamp examination, visual acuity test as well as all common cultures and stains were performed to measure the outcome. Visual acuity significantly improved from 0.2 to best-corrected visual acuity 0.7 at the 5-month postoperative visit. The cornea regained its clarity. Total re-epithelialization of the injured area was observed. It is of primary importance to remove all the debris from a cement splash at the first available opportunity. Superficial keratectomy and amniotic membrane grafting may be the best methods for the re-epithelialization and reconstruction of the ocular surface.

Adult↗