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Chia-Yang Liu

Publications and source records attributed to Chia-Yang Liu.

At least 19 recordsLinked to original sources
PubMed2006

Preservation and expansion of the primate keratocyte phenotype by downregulating TGF-beta signaling in a low-calcium, serum-free medium.

PURPOSE: To demonstrate whether the original keratocyte phenotype is maintained with proliferative activity by suppressing TGF-beta signaling in rhesus monkey keratocytes expanded in a serum-free and low-[Ca2+] medium. METHODS: Rhesus monkey keratocytes were isolated from central corneal buttons by collagenase digestion for 16 hours, seeded on plastic in Dulbecco's modified Eagle's medium (DMEM) containing insulin-transferrin-sodium selenite (ITS) supplement (DMEM/ITS) or 10% fetal bovine serum (DMEM/10% FBS), or in a defined keratinocyte serum-free medium (KSFM). After confluence, cells in KSFM were continuously subcultured at a 1-to-3 split. Cellular proliferation was analyzed by immunostaining for Ki67 and the MTT assay. The cellular phenotype was determined by immunostaining for aldehyde dehydrogenase (ALDH), keratocan, and CD34 and by the expression of keratocan promoter-driven enhanced cyan fluorescent protein (ECFP). The stability of the keratocyte phenotype was examined by switching KSFM to DMEM/ITS and DMEM/10% FBS. TGF-beta signaling was monitored by measuring the promoter activity of TGF-beta1, -beta2, and -beta RII after transient adenoviral transfection, and cytolocalization of Smad2 and Smad4. RESULTS: In KSFM, monkey keratocytes proliferated while maintaining the expression of keratocan, CD34, and ALDH proteins and keratocan promoter-driven ECFP for at least 15 passages. The nuclear accumulation of Smad2 and Smad4 and the promoter activities of TGF-beta1 and -beta RII were significantly downregulated in KSFM compared with DMEM/10% FBS. In KSFM, an increase of [Ca2+] to 1.8 mM and addition of 10% FBS synergistically downregulated the keratocan promoter activity, facilitated Smad2 and Smad4 nuclear translocation, and upregulated TGF-beta1 and -beta RII promoter activities. CONCLUSIONS: The normal monkey keratocyte phenotype can be maintained in a low-calcium, serum-free medium by downregulating Smad-mediated TGF-beta signaling.

Adenoviridae↗
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

Keratocan, a cornea-specific keratan sulfate proteoglycan, is regulated by lumican.

Lumican is an extracellular matrix glycoprotein widely distributed in mammalian connective tissues. Corneal lumican modified with keratan sulfate constitutes one of the major proteoglycans of the stroma. Lumican-null mice exhibit altered collagen fibril organization and loss of corneal transparency. A closely related protein, keratocan, carries the remaining keratan sulfate of the cornea, but keratocan-null mice exhibit a less severe corneal phenotype. In the current study, we examined the effect of lumican overexpression in corneas of wild type mice. These mice showed no alteration in collagen organization or transparency but had increased keratocan expression at both protein and mRNA levels. Corneas of lumican-null mice showed decreased keratocan. This coupling of keratocan expression with lumican also was observed after intrastromal injection of a lumican expression minigene into the corneal stroma of Lum-/- mice. Small interfering RNA knockdown of lumican in vitro reduced keratocan expression, whereas co-injection of a lumican-expressing minigene with a beta-galactosidase reporter driven by the keratocan promoter demonstrated an increase of keratocan transcriptional activity in response to lumican expression in Lum-/- corneas in vivo. These observations demonstrate that lumican has a novel regulatory role in keratocan expression at the transcriptional level. Such results help provide an explanation for the differences in severity of corneal manifestation found in Lum-/- and Kera-/- mice. The results also suggest a critical level of small proteoglycans to be essential for collagen organization but that overabundance is not detrimental to extracellular matrix morphogenesis.

Animals↗
PubMed2005

Over expression of FGF7 enhances cell proliferation but fails to cause pathology in corneal epithelium of Kerapr-rtTA/FGF7 bitransgenic mice.

PURPOSE: Available evidence suggests that fibroblast growth factor 7 (FGF7, also known as keratinocyte growth factor, KGF) serves as a paracrine growth factor modulating corneal epithelial cell proliferation. In the present study, we used a binary inducible transgenic mouse model to examine the role of FGF7 on corneal epithelium proliferation. METHODS: A keratocyte specific 3.2 kb murine keratocan promoter (Kerapr) was used to prepare Kerapr-rtTA transgenic (Kr) mice that constitutively overexpress reverse tetracycline transcription activator (rtTA) by cornea stromal keratocytes. The Kr mice were crossed with tet-O-FGF7 mice to produce Kr/tet-O-FGF7 bitransgenic mice. Expression of human FGF7 (hFGF7) was induced by the administration of doxycycline via intraperitoneal injection and/or feeding mice doxycycline in drinking water and chow. Overexpression of hFGF7 was confirmed by RT-PCR and western blot. BrdU incorporation was used to determine cell proliferation. RESULTS: The rtTA mRNA and protein were constitutively expressed by the cornea with or without doxycycline induction, whereas hFGF7 was detected only in Kr/tet-O-FGF7 bitransgenic mice upon induction by doxycycline. Examination of induction kinetics in adult Kr/tet-O-FGF7 bitransgenic mice after a single intraperitoneal injection of doxycycline revealed that hFGF7 mRNA expression was detected 12 h after doxycycline administration, peaked at 36 h, was sustained up to 48 h, and declined thereafter. The elevated level of hFGF7 expression coincided with hyperproliferation of corneal epithelial cells. In bitransgenic mice, the number of BrdU labeled cells increased after 36 and 48 h of transgene induction compared to controls of noninduced bitransgenic or doxycycline treated single transgenic mice. The BrdU labeling index was 33+/-9.2 positive cells per corneal section for Kr/tet-O-FGF7 bitransgenic mice and 25+/-9.3 for tet-O-FGF7 single transgenic mice at 36 h post-doxycycline treatment. However, the excess FGF7 driven by doxycycline induction did not produce severe perturbation of corneal epithelium homeostasis. CONCLUSIONS: Our results demonstrate that the doxycycline inducible system is effective in regulating transgene expression in corneal stroma of Kr/tet-O-FGF7 bitransgenic mice. However, the development of pathology resulting from the overexpression of transgenes may depend on whether the amount of transgene product present is sufficient to alter the homeostasis of the targeted tissues.

Animals↗
PubMed2005

Excess biglycan causes eyelid malformation by perturbing muscle development and TGF-alpha signaling.

Tissue morphogenesis during development is regulated by growth factors and cytokines, and is characterized by constant remodeling of extracellular matrix (ECM) in response to signaling molecules, for example, growth factors, cytokines, and so forth. Proteoglycans that bind growth factors are potential regulators of tissue morphogenesis during embryonic development. In this study, we showed that transgenic mice overexpressing biglycan under the keratocan promoter exhibited exposure keratitis and premature eye opening from noninfectious eyelid ulceration due to perturbation of eyelid muscle formation and the failure of meibomian gland formation. In addition, in vitro analysis revealed that biglycan binds to TGF-alpha, thus interrupting EGFR signaling pathways essential for mesenchymal cell migration induced by eyelid epithelium. The defects of TGF-alpha signaling by excess biglycan were further augmented by the interruption of the autocrine or paracrine loop of the EGFR signaling pathway of HB-EGF expression elicited by TGF-alpha. These results are consistent with the notion that under physiological conditions, biglycan secreted by mesenchymal cells serves as a regulatory molecule for the formation of a TGF-alpha gradient serving as a morphogen of eyelid morphogenesis.

Actins↗
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

Characterization of tetracycline-inducible bitransgenic Krt12rtTA/+/tet-O-LacZ mice.

PURPOSE: To prepare binary transgenic mouse lines that overexpress reporter genes in a corneal-epithelium-specific manner when induced by doxycycline. METHODS: A gene-targeting construct containing an internal ribosomal entry site-reverse tetracycline transcription activator (IRES-rtTA) cassette was inserted into the Krt12 allele (keratin 12 gene) to produce a knock-in Krt12(rtTA/+) mouse line through gene-targeting techniques. The Krt12(rtTA/+) knock-in mice were bred with tet-O-LacZ reporter mice to obtain Krt12(rtTA/+)/tet-O-LacZ bitransgenic mice. The expression of the LacZ gene was induced in bitransgenic mice by administration of doxycycline in the drinking water and chow. RESULTS: Administration of doxycycline induced a 15-fold increase of beta-galactosidase activity in the cornea of adult bitransgenic mice (Krt12(rtTA/+)/tet-O-lacZ). Administration of doxycycline either to single transgenic Krt12(rtTA/+) or tet-O-LacZ mice as a control did not induce overexpression of LacZ as it did in the bitransgenic mice. The induction of beta-galactosidase enzyme activity by doxycycline in bitransgenic mice took place in 24 hours and reached a plateau by 2 days. Histochemical analysis also showed that beta-galactosidase induction was limited to the corneal epithelium of bitransgenic mice fed doxycycline. The increased beta-galactosidase activity in corneal epithelium caused by doxycycline returned to basal levels in 4 weeks after the antibiotics were omitted from the diet. CONCLUSIONS: A binary mouse model has been successfully established that conditionally overexpresses reporter genes in corneal epithelium. This mouse model will be useful in elucidating signaling pathways of various growth factors and cytokines and gene functions in the maintenance of homeostasis and pathogenesis in the adult mouse cornea.

Animals↗
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

Fibroblast growth factor 2: roles of regulation of lens cell proliferation and epithelial-mesenchymal transition in response to injury.

PURPOSE: To examine the role of fibroblast growth factor 2 (FGF2) in regulating lens cell proliferation and epithelial-mesenchymal transition (EMT) in response to injury. METHODS: The amount of FGF2 protein was determined in healing, injured rat lenses by enzyme immunoassay. The effects of FGF2 and transforming growth factor beta2 (TGFbeta2) on cell proliferation of alphaTN4 cells (a mouse lens epithelial cell line) were determined. FGF2-knockout mice were used to further examine the role of endogenous FGF2 on injury-induced epithelial cell proliferation and EMT. The anterior lens capsule was injured by a hypodermic needle under both general and topical anesthesia in one eye of 34 fgf2+/+ mice and 42 fgf2-/- mice. At days 2, 5, and 10 post-injury the mice were sacrificed following a 2 h labeling period with bromo-deoxyuridine (BrdU). The number of BrdU-positive cells in each specimen was determined. RESULTS: A capsular break caused a 10 fold increase of FGF2 protein accumulated in rat lens 14 days after injury. Addition of 3.43 ng/ml FGF2 enhanced proliferation of alphaTN4 cells. This occurred in the presence or absence of exogenous TGFbeta2, that has an inhibitory effect on alphaTN4 cell proliferation. Significantly fewer BrdU-labeled cells were found in fgf2-/- mice than in fgf2+/+ mice during healing post-injury. However, lacking FGF2 did not alter the expression patterns of alpha-smooth muscle actin and collagen type I, markers of EMT in lens cells. CONCLUSIONS: Endogenous FGF2 is required for increased cell proliferation but not essential for EMT during the lens response to injury.

Actins↗
PubMed2004

Smad3 signaling is required for epithelial-mesenchymal transition of lens epithelium after injury.

Lens epithelial cells undergo epithelial-mesenchymal transition (EMT) after injury as in cataract extraction, leading to fibrosis of the lens capsule. Fibrosis of the anterior capsule can be modeled in the mouse by capsular injury in the lens, which results in EMT of the lens epithelium and subsequent deposition of extracellular matrix without contamination of other cell types from outside the lens. We have previously shown that signaling via Smad3, a key signal-transducing element downstream of transforming growth factor (TGF)-beta and activin receptors, is activated in lens epithelial cells by 12 hours after injury and that this Smad3 activation is blocked by administration of a TGF-beta 2-neutralizing antibody in mice. We now show that EMT of primary lens epithelial cells in vitro depends on TGF-beta expression and that injury-induced EMT in vivo depends, more specifically, on signaling via Smad3. Loss of Smad3 in mice blocks both morphological changes of lens epithelium to a mesenchymal phenotype and expression of the EMT markers snail, alpha-smooth muscle actin, lumican, and type I collagen in response to injury in vivo or to exposure to exogenous TGF-beta in organ culture. The results suggest that blocking the Smad3 pathway might be beneficial in inhibiting capsular fibrosis after injury and/or surgery.

Animals↗
PubMed2004

PITX2 gain-of-function in Rieger syndrome eye model.

The human autosomal-dominant disorder Axenfeld-Rieger syndrome presents with defects in development of the eyes, teeth, and umbilicus. The eye manifests with iris ruptures, irido-corneal adhesions, cloudy corneas, and glaucoma. Transcription factors such as PITX2 and FOXC1 have been found to carry point mutations, causing the disorder. However, for approximately 40% of the cases, the pathogenesis is unknown. It has been reported that some mutations in PITX2 increase transactivation, whereas most mutations cause defects in DNA binding or transactivation. It is not known whether up-regulation of PITX2 activity can cause the disorder as well. Here we test this hypothesis directly by overexpressing PITX2A as a transgene in mouse corneal mesenchyme and iris, using keratocan-flanking sequences. The mice presented with corneal opacification, corneal hypertrophy, irido-corneal adhesions, and severely degenerated retina, resembling glaucoma. The corneal hypertrophy also resembles the corneal hypertrophy of Pitx2-/- mice. Control transgenic mice carrying point mutations T68P or K88E in PITX2A were normal. These findings indicate a novel pathogenetic mechanism in which excess corneal and iridal PITX2A cause glaucoma and anterior defects that closely resemble Axenfeld-Rieger syndrome.

Alleles↗
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

Role of p38 MAP kinase in regulation of cell migration and proliferation in healing corneal epithelium.

PURPOSE: The purpose of the present study was to examine the roles of signaling pathways potentially activated by TGFbeta (i.e., Smad and p38 mitogen-activated kinase [MAPK]) in regulation of cell migration and proliferation of healing mouse corneal epithelium. METHODS: Activation of Smads or p38MAPK was evaluated by immunohistochemistry in healing mouse corneal epithelium after debridement. The role of endogenous TGFbeta or p38MAPK in epithelial healing was determined in organ-cultured mouse corneas with an epithelial defect, in the presence or absence of a TGFbeta-neutralizing antibody or p38MAPK inhibitors, respectively. Cell proliferation was evaluated by incorporation of bromodeoxyuridine. RESULTS: Migrating mouse corneal epithelium had minimal cell proliferation. Smad3 and -4 were found in nuclei of normal corneal epithelium, whereas they were absent in nuclei of migrating cells in association with Smad7 upregulation on epithelial debridement. Administration of TGFbeta-neutralizing antibody reduced the protein expression of Smad7 in vivo after a corneal injury. In contrast, phosphorylation and nuclear translocation of p38MAPK were markedly evident in migrating epithelium during healing, but not in uninjured epithelium. In organ culture, addition of p38MAPK inhibitors blocked cell migration more markedly than neutralizing TGFbeta-antibody and enhanced cell proliferation in the injured corneal epithelium, in association with phosphorylation of Erk. CONCLUSIONS: Endogenous TGFbeta enhances migration of corneal epithelium during wound healing in mice. The p38MAPK, but not the Smad, cascade plays a major role in promoting cell migration and in suppressing cell proliferation in migrating epithelium.

Animals↗
PubMed2004

In vivo gene delivery and visualization of corneal stromal cells using an adenoviral vector and keratocyte-specific promoter.

PURPOSE: This study was conducted to determine whether intrastromal injection of adenoviral construct could be used to transfect corneal stroma cells effectively in vivo and to determine whether a tissue-specific promoter could be used to express exogenous genes in keratocytes. METHODS: An adenoviral construct with a cytomegalovirus (pCMV)-driven enhanced green fluorescent protein (EGFP) reporter gene was injected into the stroma of murine corneas. In vivo expression was quantitated and samples were analyzed by in vivo stereomicroscopy, and ex vivo expression was determined by confocal three dimensional (3-D) reconstruction. The 3.2-kb keratocan promoter was used to drive tissue-specific reporter gene expression in vivo. RESULTS: EGFP expression was first detected in vivo 11 hours after injection of adeno-EGFP in the corneal stroma, with a duration of approximately 3 weeks. Ex vivo wholemount cornea confocal analysis with 3-D reconstruction allowed visualization of EGFP expression in corneal stroma cells, to accurately assess cellular architecture and distribution in the corneal stroma. Naked pCMV-EGFP plasmid DNA did not express the reporter gene to the levels of the adeno-EGFP. The 3.2-kb keratocan promoter was capable of driving EGFP tissue-specific expression in the cornea. CONCLUSIONS: Intrastromal injection of adenovirus packaged DNA constructs is a rapid and efficient way to deliver and express genes in the corneal stroma. Intrastromal injection is also capable of delivering tissue-specific promoter constructs to the corneal stroma for gene expression. Furthermore, 3-D reconstruction provides a powerful tool for enhanced visualization of the corneal stroma environment and cellular biology.

Adenoviridae↗
PubMed2004

Characterization of corneal pannus removed from patients with total limbal stem cell deficiency.

PURPOSE: To determine the epithelial lineage of origin in corneal pannus tissue surgically removed from patients with total limbal stem cell (SC) deficiency. METHODS: The lineage of origin of the entire conjunctivalized pannus removed from eight corneas with a diagnosis of total limbal SC deficiency was characterized by anti-keratin (K)-3 and anti-K19 monoclonal antibodies. The protein and mRNA of epithelial outgrowth from segments of five such pannus specimens were analyzed by Western blot and reverse transcription-polymerase chain reaction, respectively. RESULTS: Cross sections of all eight specimens showed a stratified epithelium with goblet cells expressing mucin (MUC)-5AC, and a stroma showing blood vessels and inflammatory cell infiltrates. Immunostaining showed full-thickness expression of K19 in the entire pannus of all eight specimens. Expression of K3 was negative in seven patients, but was sporadically positive in a patient with Stevens-Johnson syndrome. In culture, all five pannus specimens generated a compact, small epithelial cell outgrowth, and except for one, reached confluence in 2 to 3 weeks. The K3/K12 pair was expressed by extracts of cell outgrowth from the control limbal epithelial explant, but not in all five pannus specimens. A 60-kDa band of DeltaNp63 was expressed in the control specimen and in all five pannus specimens. Cell outgrowth expressed K3 transcript in three, but none showed K12 transcript. CONCLUSIONS: The resultant epithelial phenotype of the pannus tissue was not corneal, as evidenced by the negative staining to cornea-specific K12 mRNA and protein, but was conjunctival, as evidenced by the presence of goblet cells, the weak expression of K3, and the strong expression of K19. The abundant expression of DeltaNp63 in such conjunctiva-derived epithelium in eyes with total limbal SC deficiency raises doubts as to its validity as a limbal SC marker.

Adult↗
PubMed2004

CD-34 expression by cultured human keratocytes is downregulated during myofibroblast differentiation induced by TGF-beta1.

PURPOSE: To establish CD34 as a cell surface marker for human keratocytes and to demonstrate its downregulation during TGF-beta1-induced myofibroblast differentiation. METHODS: Collagenase-isolated keratocytes were seeded and subcultured on plastic or amniotic membrane matrix (AM) in DMEM, with or without 10% FBS, in serum-free DMEM containing insulin-transferrin-sodium selenite (ITS) with 10, 100, and 1000 pg/mL TGF-beta1 or in DMEM with 1% FBS and 10 ng/mL TGF-beta1. Protein expression of CD34 and alpha-smooth muscle actin (alpha-SMA) was measured by Western blot and immunostaining. RESULTS: Keratocytes, expressing CD34 in normal human corneas, continued to express CD34 when cultured on AM in serum-containing medium and on plastic in serum-free medium, but expression was lost on plastic in serum-containing medium. In serum-containing medium, expression of CD34, but not alpha-SMA, was maintained by cells continuously passaged on AM. In contrast, cells expressed alpha-SMA without CD34 when continuously passaged on plastic. Expression of alpha-SMA by cells on plastic was downregulated without CD34 when subcultured on AM. CD34 expression by cells on AM was downregulated, whereas alpha-SMA expression was upregulated when cells were subcultured on plastic. In serum-free medium, CD34 expression was maintained by cells treated with 10 pg/mL TGF-beta1, but was lost when treated with a higher concentration on plastic for 5 days. In 1% FBS, AM-expanded keratocytes rapidly became alpha-SMA-expressing myofibroblasts if subpassaged on plastic and treated with 10 ng/mL TGF-beta1, but failed to do so if cultured on AM, even for 7 days. CONCLUSIONS: These findings indicate that CD34 is expressed by human keratocytes in vivo and in vitro. Myofibroblast differentiation promoted by TGF-beta1 downregulates CD34 expression. Maintenance of CD34 expression by AM is consistent with a reported effect of AM on suppressing TGF-beta signaling.

Actins↗