Biomedical subjects

Edgar M Espana

Publications and source records attributed to Edgar M Espana.

At least 19 recordsLinked to original sources

PubMed2006

A newly designed glaucoma drainage implant made of poly(styrene-b-isobutylene-b-styrene): biocompatibility and function in normal rabbit eyes.

OBJECTIVE: To report clinical evaluation, flow patency, and histopathological findings of a novel glaucoma drainage implant (GDI) made of poly(styrene-b-isobutylene-b-styrene) (SIBS) in rabbits. METHODS: In 16 normal eyes, the proximal end of the SIBS GDI was inserted into the anterior chamber while the distal end was placed in the subconjunctival space. A control group underwent implantation of a similarly designed silicone GDI. Slitlamp follow-up and intraocular pressure measurements were recorded. Flow patency was evaluated by injecting 0.01% fluorescein into the anterior chamber. Immunostaining against collagen IV, macrophages, and alpha smooth muscle actin was performed. RESULTS: Slitlamp examination suggested adequate biocompatibility. A low and diffuse bleb was observed in the SIBS group. All SIBS tubes were patent 6 months after insertion. Immunostaining demonstrated noncontinuous collagen deposition. No macrophages or myofibroblasts were visible around the SIBS tubes. In contrast, silicone induced collagen deposition and myofibroblast differentiation. CONCLUSION: This new GDI is clinically biocompatible in the rabbit and maintained 100% patency at 6 months. A remarkable difference was the absence of myofibroblasts in the surrounding tissue in the SIBS group. CLINICAL RELEVANCE: This novel GDI made of SIBS would prevent the feared complication of hypotony and will decrease the amount of subconjunctival fibrosis.

Actins↗

PubMed2006

Corneal stroma regeneration in felines after supradescemetic keratoprosthesis implantation.

PURPOSE: To show corneal regeneration in 3 cats that underwent lamellar keratectomy (90%) depth during supradescemetic keratoprosthetic implantation. METHODS: Three 2-year-old cats that underwent spontaneous keratoprosthesis extrusion between 15 and 150 days after implanting a supradescemetic prosthesis into their right eyes were studied. Corneal structures and stroma thickness were evaluated by slit-lamp photographs, pachymetry, and confocal microscopy. Regenerated corneal epithelial cells, stroma matrix, and keratocyte morphology were studied with histology and transmission electron microscopy. Epithelial and stromal cell immunocharacterization was performed. RESULTS: Corneas progressively regained normal thickness and improved clarity within 40 to 60 days. Slit-lamp photographs and pachymetry showed gains in stromal thickness until 600 microm or more. In vivo confocal microscopy showed the restoration of normal epithelium and stroma in all cats. Corneal nerves were seen in the regenerated stroma of 2 cats. Immunostaining showed absent alpha-smooth muscle actin (SMA) expression and a keratin K3-expressing epithelium. Electron microscopy showed regeneration of normal epithelium with a well-formed basement membrane, organized corneal lamellae, and the presence of normal keratocytes. CONCLUSION: Felines are capable of regenerating corneal structures including epithelium and reinnervated stroma matrix after deep lamellar keratectomy. The use of feline models in corneal keratoprosthesis is therefore questionable.

Actins↗

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

Validation of the Barraquer tonometer for high intraocular pressure estimation.

PURPOSE: To compare the pneumatonometer and the Tono-Pen XL in a closed ex-vivo system in human eye bank eyes at high intraocular pressures (IOP) and evaluate the validity of high IOP measurements with the Barraquer tonometer. METHODS: Intraocular pressure was monitored by cannulation of the anterior chamber and vitreous cavity in eight human cadaver eyes (mean donor age: 77.3 +/- 4.9 years, range: 72 to 84 years). Intraocular pressure measurements were taken at 50, 65, and 90 mmHg with the Tono-Pen XL and pneumatonometer. Intraocular pressure was raised to 110 mmHg and then the eyes were deflated slowly until they reached 50 mmHg. Pressure readings with the Barraquer tonometer were recorded when the corneal tonometer interface reached the inner and outer rings. RESULTS: The Tono-Pen XL underestimated IOP, a tendency that was more evident at higher IOP In contrast, the pneumatonometer was more accurate and reliable at IOP of 50 and 65 mmHg but its readings underestimated IOP at 90 mmHg. The Barraquer tonometer used in this experiment accurately estimated high IOP A variability of 5.9 mmHg and 5.8 mmHg were recorded for the inner and outer ring, respectively. CONCLUSIONS: The Tono-pen XL is an inadequate instrument to assess pressures normally encountered during LASIK flap creation in an ex vivo model using human cadaver eyes. The pneumatonometer and the Barraquer tonometer are accurate instruments at high IOP; however, the pneumatonometer underestimated pressures around 90 mmHg.

Aged↗

PubMed2005

Amniotic membrane induces apoptosis of interferon-gamma activated macrophages in vitro.

Amniotic membrane (AM) used as a temporary or permanent graft for ocular surface reconstruction has a potent anti-inflammatory effect. We would like to investigate the mechanism whereby AM induces macrophage apoptosis in vitro. Mouse macrophages, Raw 264.7 cells, were cultured on plastic, type I collagen, corneal stromal slice or AM stromal matrix in serum-free medium with or without interferon-gamma (IFN-gamma). Cells were stained by LIVE/DEAD assay, Hoechst-33342, and TUNEL assay for cell death and apoptosis. Cell lysates and conditioned media were analysed by Cell Death Detection ELISA assay for quantitation of apoptosis. Conditioned media were also analysed by Griess assay for the nitrite concentration and ELISA assay for tumour necrosis factor alpha (TNF-alpha) concentration. Lysates of cells were subjected to Western blot analyses of IKK-alpha, IKK-beta, p65 (RelA) subunit of nuclear factor kappaB (NF-kappaB), total Akt, phospho-Akt (Ser473), and phospho-FKHR (Thr24)/phosphor-FKHRL1 (Thr32). At 48hr after cultivation, cells showed a low level of apoptosis when cultured on plastic, type I collagen and corneal stromal slice with or without IFN-gamma and on AM without IFN-gamma. Nevertheless, cells showed a significant increase of apoptosis when cultured on AM with IFN-gamma activation, and this phenomenon became apparent only after 48 hr. IFN-gamma-activated macrophages on plastic continuously produced nitric oxide (NO) and TNF-alpha during 72 hr culturing. In contrast, there was no NO and TNF-alpha production after 48 hr culture on AM. NO inhibitors, L-NMMA and L-NIL, attenuated NO production of IFN-gamma-activated macrophages on AM, while apoptosis was not decreased accordingly. Expression of IKK-alpha, IKK-beta, p65 (RelA) subunit of NF-kappaB total Akt, phosopho-Akt (Ser473), and phospho-FKHR (Thr24)/FKHRL1 (Thr32) was all down-regulated in IFN-gamma-activated macrophages cultured on AM. In conclusion, AM stromal matrix induces apoptosis of IFN-gamma activated, but not non-activated macrophages, not through the generation of NO, but instead by down-regulating anti-apoptotic NF-kappaB and Akt-FKHR signalling pathways.

Amnion↗

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

Amniotic membrane maintains the phenotype of rabbit retinal pigment epithelial cells in culture.

The success of surgical removal of choroidal neovascularisation followed by transplantation of autologous retinal pigment epithelial cells (RPE) for age-related macular degeneration (ARMD) may be limited by damage in Bruch's membrane. We investigated whether amniotic membrane (AM) might be used as an alternative basement membrane-containing matrix to support RPE growth and differentiation. Primary RPE plastic cultures were established from freshly enucleated Dutch belted rabbit eyes in DMEM/F12 containing 0.1 mM Ca(++) and 10% dialysed FBS. Upon subconfluence, cells were subcultured at 5000-9000 cells cm(-2) in the above-mentioned culture medium on intact AM (iAM), epithelially denuded AM (dAM) or plastic. After confluence, the Ca(++) concentration in the medium was increased to 1.8 mm for 4 weeks. Growth and morphology were monitored by phase contrast microscopy, and the phenotype by immunostaining with antibodies against cytokeratin 18, tight junction protein ZO-1, and RPE65 protein, and by transepithelial resistance (TER) measurement. Immunostaining to cytokeratin 18 confirmed the epithelial origin of isolated cells in both primary culture and subcultures. Compared to plastic cultures, RPE increased pigmentation within 24 hr after seeding on AM, with iAM being more pronounced than dAM. RPE adopted a hexagonal epithelial phenotype with more organised pigmentation, strong expression of ZO-1 and RPE65, and a significantly higher TER 4 weeks after Ca(++) switch on dAM. Our results indicate that AM may be used as a basement membrane-containing matrix to maintain RPE phenotype in vitro, and may facilitate subsequent transplantation to treat ARMD.

Amnion↗

PubMed2005

Correlation of corneal complications with eyelid cicatricial pathologies in patients with Stevens-Johnson syndrome and toxic epidermal necrolysis syndrome.

PURPOSE: To look at the correlation between many factors (time of hospitalization, floppy eyelid syndrome, trichiasis, open lacrimal puncta, symblepharon, and aqueous tear deficiency) and corneal complications in Stevens-Johnson syndrome (SJS). DESIGN: Observational cases series. PATIENTS: Clinical data were retrospectively reviewed from 38 patients (32.7+/-20.1 years old) with SJS (n = 11) and with toxic epidermal necrolysis (TENS) (n = 27) from January 2002 to August 2004. One case report with SJS was included to verify the presence of tarsal/lid margin ulceration at the acute stage. METHODS: The medical history was retrieved regarding presumed causative medications used within 15 days and the duration of hospitalization. Data of the latest photographic documentation and eye examination were compared and correlated in a masked fashion. MAIN OUTCOME MEASURES: Floppy eyelid, trichiasis, lid margin keratinization, meibomian gland orifice metaplasia, symblepharon, tarsal scar, and corneal complications. RESULTS: Acute SJS/TENS was characterized by tarsal conjunctival ulceration. Keratinization of the eyelid margin with variable degrees of meibomian gland dysfunction was observed in all cases. Floppy eyelid, trichiasis, partially or totally opened lacrimal punctum, symblepharon, and aqueous tear deficiency were not significantly correlated with corneal complications. In contrast, there was a strong correlation between the severity of eyelid margin and tarsal pathology and the extent of corneal complications (Spearman r, 0.54; P = 0.0005). A multivariable regression analysis also showed that the extent of eyelid and tarsal pathology had a significant effect on corneal complications (coefficient, 0.84; P = 0.006). CONCLUSIONS: Patients with acute SJS/TENS are characterized by severe inflammation and ulceration of the tarsal conjunctiva and lid margins. If left unattended, lid margin keratinization and tarsal scar, together with lipid tear deficiency, contribute to corneal complications because of blink-related microtrauma. Attempts to suppress inflammation and scarring by amniotic membrane transplantation at the acute stage and to prevent microtrauma at the chronic stage are vital to avoid sight-threatening complications.

Adolescent↗

PubMed2005

Intrastromal invasion by limbal epithelial cells is mediated by epithelial-mesenchymal transition activated by air exposure.

Corneal epithelial stem cells are located in the basal layer of the limbus between the cornea and the conjunctiva. Regulation of these limbal epithelial progenitor cells by the stromal niche dictates corneal surface health. To further characterize this process, limbal explants were cultured at the air-fluid interface, termed air-lifting, to stimulate the niche. As compared to submerged cultures, air-lifting significantly promoted epithelial stratification, migration, proliferation, and intrastromal invasion by limbal epithelial cells. Epithelial intrastromal invasion was noted when the limbal, but not corneal, epithelium was recombined with the limbal stroma containing live, but not dead, cells. Invading limbal basal cells displayed up-regulated nuclear expression of p63 and Ki67, down-regulated E-cadherin and cornea-specific keratin 3, and switched expression of beta-catenin from intercellular junctions to the nucleus and cytoplasm, indicating the activation of the Wnt/beta-catenin pathway. Invaded cells isolated by collagenase from the stroma of air-lifted, but not submerged, explants showed vivid clonal growth on 3T3 fibroblast feeder layers and complete epithelial-mesenchymal transition by expressing nuclear p63 and cytoplasmic S100A4. These findings collectively suggest that epithelial-mesenchymal transition via the Wnt/beta-catenin pathway influences the fate of limbal epithelial cells, likely to be progenitor cells, between regeneration and fibrosis when the stromal niche is activated.

Air↗

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

A case of conjunctiva-cornea intraepithelial neoplasia successfully treated with topical mitomycin C and interferon alfa-2b in cycles.

PURPOSE: To report a case of conjunctiva-cornea intraepithelial neoplasia (CCIN) treated with topical mitomycin C (MMC) and interferon alfa-2b in cycles. METHODS: A 52-year-old woman referred for limbal stem cell deficiency and epithelial defect was found instead to have CCIN. The diagnosis was confirmed by impression cytology and treated in cycles with topical 0.02% MMC for 14 days in the first cycle, 12 days in the second cycle, and 3 days in the third cycle followed by topical interferon alfa-2b 1 x 106 U/mL for 11 days. RESULT: Topical MMC for 2.5 cycles resulted in 95% reduction of CCIN and improvement of 2 lines of vision. Because of intolerance to MMC, she was switched to topical interferon alfa-2b for 11 days, resulting in total resolution of CCIN, which lasted for at least 10 months. CONCLUSIONS: After differentiation from a persistent corneal epithelial defect and limbal stem cell deficiency by dye staining and impression cytology, the patient in this case of CCIN was successfully treated with topical MMC and interferon alfa-2b in cycles.

Administration, Topical↗

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↗

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↗

PubMed2003

Amniotic membrane transplantation for reconstruction of the conjunctival fornices.

PURPOSE: To describe the clinical outcome of amniotic membrane transplantation (AMT) for fornix reconstruction in a variety of ocular surface disorders. DESIGN: Noncomparative interventional case series. PARTICIPANTS: Seventeen eyes in 15 patients with symblepharon. Four eyes had ocular-cicatricial pemphigoid, two eyes had symblepharon after pterygium excision, four eyes had chemical or mechanical trauma, two eyes had strabismus surgery, two eyes (one patient) had Stevens-Johnson syndrome, one eye had toxic epidermal necrolysis, and two eyes (one patient) had chronic allergic conjunctivitis. INTERVENTION: The subconjunctival scar tissue was dissected from the episclera, and the freed conjunctival flap was recessed to the fornix. A layer of amniotic membrane (AM) was applied to cover the exposed episclera. The fornical edge of the membrane was anchored with sutures passing through the full thickness of the lid. MAIN OUTCOME MEASURES: A deep conjunctival fornix, lack of motility restriction. RESULTS: The mean follow-up period was 37 +/- 24 months (range, 9-84 months). Complete fornix reconstruction was demonstrated in 12 of 17 eyes (70.6%), whereas 2 eyes had a partial success, and 3 eyes (3 patients) had recurrence of symblepharon with restricted motility. In eyes that demonstrated partial success or failure, the underlying etiology was either an autoimmune disorder or a recurrent pterygium. The most successful outcome was observed in eyes with symblepharon associated with trauma. CONCLUSIONS: AMT is an effective method of fornix reconstruction for the repair of symblepharon in a variety of ocular surface disorders. Future modifications, including an epithelial cellular component on the AM (conjunctival autograft or ex vivo expanded epithelial stem cells) may improve the outcome of this surgical procedure.

Adult↗

PubMed2003

Phenotypic study of a case receiving a keratolimbal allograft and amniotic membrane for total limbal stem cell deficiency.

PURPOSE: To report the expression pattern of key molecules by the reconstructed corneal epithelium after a keratolimbal allograft (KLAL) and amniotic membrane transplantation (AMT) for total limbal stem cell deficiency. DESIGN: Interventional case report. METHOD: A 50-year-old woman with severe chemical burns in both eyes received an AMT as a temporary patch at the acute stage, and a KLAL with AMT as a graft at the chronic stage for total limbal stem cell deficiency. The corneal button removed during subsequent corneal transplantation was submitted for immunofluorescence staining with monoclonal antibodies against keratin K3, MUC5AC, connexin 43, integrins alpha3beta1 and alpha6beta4, and laminin 5 for comparison with a normal cornea. RESULTS: Histologically, a normal stratified corneal epithelium has five to six cell layers that lay on the thick amniotic membrane basement membrane. The phenotype was of a corneal origin, based on expression of positive keratin K3, negative MUC5AC, and positive connexin 43. Furthermore, intact basement membrane complexes were present, evidenced by positive staining to integrins alpha3beta1 and alpha6beta4 and to laminin 5. CONCLUSIONS: A normal corneal epithelial phenotype with normal basement membrane complexes was restored after a KLAL and AMT in a case with total limbal stem cell deficiency.

Amnion↗