Biomedical subjects

James D Zieske

Publications and source records attributed to James D Zieske.

11 recordsLinked to original sources

PubMed2004

Transduction of functionally active TAT fusion proteins into cornea.

A technology has recently been developed that allows for the rapid transduction of full-length functionally active proteins into intact tissue through intravenous injection and into cultured cells. This technology involves the fusion of an 11 amino acid sequence of the HIV TAT protein to the protein of interest. In the current investigation, we determined whether functionally active TAT fusion proteins could be transduced into intact corneas by topical application. TAT-beta-galactosidase was purified from bacterial cells and applied in serial dilutions (12.5-250 nm) to cultured epithelial cells for 5 or 15 min. In addition, enucleated globes and excised corneas with or without a central 3-mm epithelial debridement were incubated with TAT-beta-galactosidase for 1 or 2 hr. Excised corneas were allowed to heal in organ culture. Transduction of active beta-galactosidase was detected by incubating the cells or corneas with X-gal. TAT-beta-galactosidase was transduced into nearly all cultured cells in a concentration-dependent manner. When TAT-beta-galactosidase was topically applied to intact corneas, only the most superficial layer of epithelium was highly transduced. When the superficial layer was removed with nitrocellulose, two to four layers of cells were transduced. In corneas with a central debridement, epithelial cells at the edge of the debridement were transduced as well as the stromal cells subjacent to the debridement. Active beta-galactosidase was maintained at least 1 day in organ culture. No X-gal reaction was seen in either cells or corneas not incubated with TAT-beta-galactosidase. Functionally active proteins can be efficiently transduced into corneal epithelial and stromal cells using TAT fusion protein technology. The intact epithelium provides a barrier to penetration of TAT proteins. This barrier can be overcome by disrupting the epithelium. TAT-mediated protein transduction may be extremely useful in studies of corneal wound healing and homeostasis.

Animals↗

PubMed2004

Cell cycle regulators at the ocular surface.

The ocular surface provides an outstanding model to examine the regulation of the proliferative cell cycle. Cells within the cornea and conjunctiva exhibit a wide range of proliferative abilities ranging from the rapidly proliferating cells in the basal cell layer of the epithelium to the quiescent keratocytes and endothelial cells. In this review, dedicated to Dr David Maurice, we will discuss four families of proteins known to regulate the cell cycle. These families include: (1) the cyclins; (2) the CIP/KIP family of cell cycle inhibitors--consisting of p21, p27, and p57; (3) the INK4 family of cell cycle inhibitors--including p16, p15, p18, and p19; and (4) the retinoblastoma family--consisting of pRb, p107, and p130. Members of all of these families have been localized in various cells in the ocular surface. We will discuss how these proteins are involved in regulating cell proliferation both in normal homeostasis and during wound healing.

Cell Cycle↗

PubMed2004

TAT-mediated protein transduction into human corneal epithelial cells: p15(INK4b) inhibits cell proliferation and stimulates cell migration.

PURPOSE: The cell cycle inhibitor p15(INK4b) has been localized in migrating corneal epithelial cells. In this study, TAT-fusion protein technology was used to transduce p15(INK4b) into human corneal epithelial cells to examine the effect on cell proliferation and migration. METHODS: Human p15(INK4b), obtained by RT-PCR, was cloned into a TAT-HA vector, and the fusion protein was purified from bacteria transformed with the TAT-HA-p15 construct. Various dilutions of TAT-HA-p15 were applied to primary human corneal epithelial cells to test potency. In addition, the effect of exposure time was examined. Cells were labeled with bromodeoxyuridine to detect proliferation, and indirect immunofluorescence was performed. Ki67 expression was also examined. To assay cell migration, human corneal epithelial cells were plated inside a cylinder and exposed to TAT-HA-p15. The cylinder was removed, the cells were allowed to spread for 2 days, and the area of cell coverage was calculated. TAT-HA-beta-galactosidase served as the control in all experiments. Finally, the extent of retinoblastoma protein phosphorylation was assayed by Western blot in cells cultured with and without TAT-HA-p15. RESULTS: TAT-HA-p15 was successfully transduced into primary human corneal epithelial cells. TAT-HA-p15 decreased proliferation in a concentration- and time-dependent manner. The migration assay showed that TAT-HA-p15 stimulated cell migration 1.8-fold. TAT-HA-beta-galactosidase had no effect on proliferation or migration. Finally, TAT-HA-p15 decreased the level of phosphorylated retinoblastoma protein by 4.9-fold. CONCLUSIONS: Active p15(INK4b) can be efficiently transduced into primary human corneal epithelial cells using TAT-fusion protein technology. p15(INK4b) appears to be sufficient to inhibit corneal epithelial cell proliferation and to stimulate cell migration.

Base Sequence↗

PubMed2004

Corneal development associated with eyelid opening.

The development of the cornea as a tissue initiates as early as five weeks in the human embryo. This development continues gradually until the time of eyelid opening, which is associated with major developmental changes. These events, most easily observed in rodents, which are born with closed eyelids, include alterations in the rate of cell proliferation in the epithelium, stroma and endothelium; differentiation of the epithelium; appearance of a tear film and tear-film-associated proteins; and swelling and thinning of the stroma. Eyelid opening is also associated with numerous alterations in gene expression. These events are the subject of this review. Readers are directed to the article by Wolosin et al., also in this volume, for an in-depth discussion of early corneal development.

Animals↗

PubMed2003

Apoptosis, necrosis, proliferation, and myofibroblast generation in the stroma following LASIK and PRK.

The aim of this study was to semi-quantitatively analyze stromal cell apoptosis, stromal cell proliferation, and myofibroblast generation over time points from 4hr to 3 months in rabbit eyes having photorefractive keratectomy (PRK) or laser in situ keratomeliusis (LASIK). Stromal cell necrosis and inflammatory cell infiltration were also studied. PRK for low myopia (-4.5diopters [D]), PRK for high myopia (-9.0D), and LASIK for high myopia (-9.0D) were performed in rabbit eyes, and corneas were obtained for examination at 4, 24, and 72hr, 1 and 4 weeks, and 3 months after surgery. A total of 144 rabbits were included in the study. Stromal cell apoptosis, proliferation, and myofibroblast generation were evaluated semi-quantitatively by TUNEL assay, immunocytochemical analysis of Ki67, and immunocytochemical analysis of alpha-smooth muscle actin, respectively. Stromal cell necrosis and characteristics of other cell types in the stroma were evaluated by electron microscopy. Keratocyte apoptosis and the subsequent proliferation and generation of myofibroblasts were qualitatively and quantitatively different in PRK for high myopia compared to either PRK for low myopia or LASIK for high myopia. Stromal cell necrosis becomes a significant form of cell death by 24hr after injury and may involve corneal fibroblasts, myofibroblasts, and inflammatory cells. Large numbers of polymorphonuclear cells and monocytes invade the cornea by 24hr after surgery and persist for over 1 week. The qualitative and quantitative differences in the cellular wound healing response after PRK for high and low myopia and LASIK for high myopia are likely determinants of the clinical differences in refractive outcome and some of the complications, such as regression and haze, seen after these procedures.

Animals↗

PubMed2003

Effect of ectopic epithelial tissue within the stroma on keratocyte apoptosis, mitosis, and myofibroblast transformation.

The purpose of this study was to examine the effects of the epithelium on processes involved in stromal wound healing. Lamellar epithelial-stromal flaps were produced in rabbit corneas with a microkeratome. Peripheral corneal epithelial tissue, central corneal epithelial tissue, or no epithelial tissue (control) was introduced beneath the flap. Corneas were removed at time points from 4 hr to 1 month after surgery. Tissue sections were analyzed with immunocytochemistry for Keratin 3 (K3) to detect epithelial antigen, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling (TUNEL) assay to detect apoptosis, immunocytochemistry for Ki67 to detect cell proliferation, and immunocytochemistry for alpha-smooth muscle actin (SMA) to detect myofibroblasts. K3 was detected at the level of the interface from 4 hr to 1 month after surgery in corneas in which epithelial tissue was introduced, but not control corneas, with the exception of one that developed epithelial in growth. Keratocyte apoptosis was significantly higher at 4 hr after flap formation in both groups in which corneal epithelial tissue was introduced beneath the flap compared with controls. Keratocyte proliferation was significantly greater at 72 hr in corneas in which epithelial tissue was introduced beneath the flap compared to the controls. Corneas in which epithelial tissue was introduced into the interface, but not control corneas, had stromal cells expressing alpha-SMA in the stroma anterior and posterior to the interface at 1 week and 1 month after surgery. This was also noted in the control cornea in which there was epithelial ingrowth. Signals derived from the corneal epithelium promote keratocyte apoptosis. Keratocyte proliferation is higher in corneas that have lamellar surgery when epithelial tissue is introduced into the interface. Epithelium-derived signals also participate in the generation and/or maintenance of myofibroblasts in the corneal stroma.

Actins↗

PubMed2003

Human diabetic corneas preserve wound healing, basement membrane, integrin and MMP-10 differences from normal corneas in organ culture.

The authors have previously documented decreased epithelial basement membrane (BM) components and alpha3beta1 epithelial integrin, and increased expression of matrix metalloproteinase (MMP)-10 in corneas of patients with diabetic retinopathy (DR) compared to normal corneas. The purpose of this study was to examine if organ-cultured DR corneas exhibited the same alterations in wound healing and diabetic marker distribution as the autopsy DR corneas. Twenty normal and 17 DR corneas were organ-cultured in serum-free medium over agar-collagen gel at the air-liquid interface for up to 45 days. Circular 5 mm central epithelial wounds were made with n-heptanol, the procedure that will preserve fragile diabetic corneal BM. Wound healing was monitored microscopically every 12 hr. Distribution of diabetic corneal epithelial markers including laminin-10 alpha5 chain, nidogen-1/entactin, integrin alpha3beta1, and MMP-10, was examined by immunofluorescence. Normal corneas healed the central epithelial defect within 3 days (mean=2.3 days), whereas DR corneas on average healed about two times slower (mean=4.5 days). In wounded and completely healed organ-cultured corneas, the patterns of studied markers were the same as in the unwounded organ-cultured corneas. This concerned both normal and DR corneas. As in vivo, normal organ-cultured corneas had continuous staining for laminin-10 and nidogen-1/entactin in the epithelial BM, strong and homogeneous staining for both chains of alpha3beta1 integrin in epithelial cells, and little if any staining for MMP-10. Organ-cultured DR corneas also had marker patterns specific for in vivo DR corneas: interrupted to no staining for laminin-10 and nidogen-1/entactin in the epithelial BM, areas of weak or disorganized alpha3beta1 integrin in epithelial cells, and significant MMP-10 staining in the epithelium and keratocytes. Fibrotic extracellular matrix and myofibroblast markers were largely absent. Thus, epithelial wound healing was much slower in organ-cultured DR corneas than in normal corneas, in complete accordance with clinical data in diabetic patients. DR corneas in organ culture preserved the same marker abnormalities as in vivo. The marker distribution was unchanged in wounded and healed organ-cultured corneas, compared to unwounded corneas. The established corneal organ culture provides an adequate system for elucidating mechanisms of epithelial alterations in human DR corneas.

Aged↗

PubMed2003

Molecular mechanisms controlling the fibrotic repair phenotype in cornea: implications for surgical outcomes.

PURPOSE: Incisional or ablation injury to the corneal stroma is repaired by deposition of a fibrotic tissue produced by activated keratocytes, whereas cells lost from the underlying stroma after epithelial abrasion are simply replaced by keratocyte replication without expression of fibrotic markers. The purpose of this study was to investigate mechanisms that determine this differential keratocyte response. METHODS: A penetrating keratectomy rabbit model was adapted for mice to study the fibrotic repair response. A mouse epithelial abrasion model was applied to study the stromal cell replacement response. A primary rabbit corneal cell culture model and an organotypic culture model were also used. RESULTS: When the epithelium was prevented from resurfacing the cornea after penetrating keratectomy, expression of fibrotic markers was considerably reduced. TGF-beta2 was determined to be a major substance produced by corneal epithelial cells capable of inducing the fibrotic phenotype. In the intact mouse cornea, TGF-beta2 was confined to the uninjured epithelium, but was released into the stroma during fibrotic repair. By contrast, TGF-beta1 was never found in the epithelium. When epithelial cells were cultured on a basement-membrane-like gel or allowed to deposit their own basement membrane in organotypic culture, TGF-beta2 production was reduced. Return of a basement membrane after wounding in vivo correlated with loss of the fibrotic phenotype. In the epithelial debridement injury model in which the basement membrane was left intact, TGF-beta2 remained confined to the corneal epithelium, consistent with the absence of a fibrotic phenotype. CONCLUSIONS: These data suggest that integrity of the basement membrane is a deciding factor in determining the regenerative character of corneal repair.

Actins↗

PubMed2002

Changes in connexin43 in early ocular surface development.

PURPOSE: In the limbo-corneal epithelium the stem and early precursor epithelial cell pool is confined to the limbal rim. Among the features associated with this spatial segregation is the general paucity of connexin43 (Cx43) within the limbal basal cell population and its complete absence in resident stem cells. The limbo-corneal epithelial lineage derives from a Cx43-positive (Cx43+) embryonic outer ectoderm. Accordingly, as a means of identifying the process through which limbal cell phenotypes emerge, we investigated the expression of Cx43 in the ocular surface of embryonic rats. METHODS: Ocular surface expression of Cx43 or K12 was determined in cryostat sections of rat embryos and eyes using immunohistological methods. RESULTS: Changes in Cx43 expression revealed the early phenotypic divergence of three main epithelial cell phenotypes of the ocular surface. An analysis of the level and distribution pattern of Cx43 puncta lead to the identification of two distinct domains by embryonic day 10 (E10), a stage that occurs soon after formation of the lens vesicle. Additionally, at E12, ectodermal cells directly adjacent to the edges of the developing retina no longer express connexin. A comparison of anatomical and expression changes throughout embryonic development demonstrated that the two early zones represent the rudiments for the epithelia of the central cornea and conjunctiva, respectively, and that the isolated Cx43-negative (Cx43-) cells represent the precursors of the basal and, putatively, stem cells of the limbal epithelium. CONCLUSIONS: Changes in Cx43 expression revealed that the phenotypic divergence of ocular surface epithelial cells and the generation of limbo-corneal stem cell precursors takes place at a very early stage in ocular development, ahead of the establishment of any identifiable anatomical or differentiation features for these domains.

Animals↗

PubMed2002

Extracellular matrix and matrix metalloproteinase changes in human corneas after complicated laser-assisted in situ keratomileusis (LASIK).

PURPOSE: To characterize extracellular matrix (ECM) and nine matrix metalloproteinase (MMP) changes in two corneas that underwent a complicated laser-assisted in situ keratomileusis (LASIK) procedure. METHODS: The first patient underwent bilateral LASIK. The flap on the left eye was transected in several locations and placed back. This cornea later developed edema, and the removed flap was analyzed after lamellar keratoplasty. The second patient had a LASIK flap lifted, replaced twice, and then completely removed. The epithelium grew over the stroma, but haze and severe ectasia occurred. After penetrating keratoplasty, the recipient cornea was analyzed. An autopsy cornea from a person who underwent uneventful LASIK and ten normal autopsy corneas served as controls. Corneas were analyzed by immunohistochemistry. RESULTS: Both flap regions in the treated corneas had marked alterations of ECM components and MMPs. Stromal deposits of various ECM proteins, including those normally absent in the central cornea (tenascin-C, fibrillin-1, type XIV collagen), were found. Rare myofibroblasts and inflammatory cells were present. The epithelial basement membrane (BM) was altered in both cases. The most dramatic change was poor or no staining for alpha3-alpha6 type IV collagen chains and thrombospondin. The limbal alpha1-alpha2 type IV collagen and laminin-2 (alpha2beta1gamma1) appeared in the central epithelial BM. Other components were altered to a lesser extent. The anterior stroma was positive for MMP-1 and MMP-2, and some MMP-7 was seen in the epithelium. These ECM and MMP patterns were not seen in uneventful LASIK or normal corneas. CONCLUSIONS: In the flaps of LASIK-treated corneas, fibrosed areas of anterior stroma had increased levels of MMP-1 and MMP-2 that may have caused loss of specific type IV collagen isoforms in the epithelial BM. These changes may reflect an ongoing wound healing process and contribute to the development of ectasia.

Adult↗

PubMedDate not supplied

Evolution, techniques, clinical outcomes, and pathophysiology of LASEK: review of the literature.

Laser subepithelial keratomileusis (LASEK) is a relatively new laser surgical procedure that combines certain elements of both laser in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK) to improve the risk/benefit ratio. Diluted alcohol is used to loosen the epithelial adhesion to the corneal stroma. The loosened epithelium is moved aside from the treatment zone as a hinged sheet. Laser ablation of the subepithelial stroma is performed before the epithelial sheet is returned to its original position. We reviewed the literature regarding modifications of the technique, indications, outcomes, and complications, as well as wound healing after LASEK surgery. This literature review of 1,421 LASEK-treated eyes provided many findings: 1) The long-term stable results in the absence of serious complications, such as infections, recurrent erosions, scars, or late-onset corneal haze formation in patients re-examined up to 5 years after LASEK; 2) Epithelial closure with recovery of functional vision was completed at days 4 to 7 in most cases; 3) A tendency toward overcorrection with PRK nomograms; 4) We hypothesize that this tendency may be due to the decreased wound healing response, which may lead to myopic regression in PRK; and 5) Postoperative discomfort and prolonged visual recovery until the epithelium closes remain the biggest disadvantages of LASEK compared to LASIK. LASEK surgery is especially valuable in patients with thin corneas who would not qualify for LASIK surgery. However, a potential superiority of LASEK to LASIK in wavefront guided ablations still remains speculative.

Humans↗