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University of Cincinnati

Department of Ophthalmology

Winston Kao, Ph.D.

We have demonstrated that transplantation of human umbilical mesenchymal stem cells cures the thin and cloudy corneas of lumican knockout mice. The treatment strategy has been expanded to treat acquired corneal diseases, e.g., alkali-burned corneas, limbal deficiency (loss of corneal stem cells) in experimental mouse models. Our preliminary results indicate that MSC transplantation prevents corneal cloudiness and ulceration caused by alkali burn. The MSC transplantation also allows the regeneration of corneal stem/progenitor cells after limbal epithelium debridement. In search of other stem cells sources besides limbus, we have cultured stem cells from hair follicles that were able to differentiate into corneal epithelial cells when the cultivated stem cells were grafted onto the denuded corneal surface of total limbal deficiency. In attempt to examine the molecular and cellular mechanism of dry eye, a disease affecting millions of American, we have created new genetically modified mouse lines that manifest malformation of Meibomian glands, the lipid secreting gland essential for maintaining healthy eyes. These new mouse lines will allow us to determine how Meibomian glands are formed during development and affected by aging.

PROGRESS REPORT

1. Molecular Analysis and Characterization of Zebrafish Lumican (zLum) gene

The lumican gene (Lum), which encodes one of the major keratan sulfate proteoglycans (KSPGs) in the vertebrate cornea and sclera, has been linked to axial myopia in humans. In this study, we chose zebrafish (Danio rerio) as an animal model to elucidate the role of lumican in the development of axial myopia. Transgenic zebrafish harboring an Enhanced Green Fluorescence Protein (EGFP) reporter gene construct downstream of a 1.7 kb zLum 5’-flanking region displayed EGFP expression in the cornea and sclera, as well as throughout the body. Down-regulation of zLum expression by antisense zLum morpholinos (MO) manifested ocular enlargement resembling human axial myopia due to disruption of the collagen fibril arrangement in the sclera and resulted in scleral thinning. Administration of muscarinic receptor antagonists, e.g., atropine, pirenzepine, effectively subdued the ocular enlargement caused by morpholinos using in vivo zebrafish larvae assays. The observation suggests that zebrafish can be used as an in vivo model for screening compounds in treating myopia. (JBC in Press)

2. Requirement of Lumican for Neutrophil Extravasation during Corneal Wound Healing

An important aspect of wound healing is the recruitment of neutrophils to the site of infection or tissue injury. Lumican, an extracellular matrix component belonging to the small leucine rich proteoglycan (SLRP) family, is one of the major keratan sulfate proteoglycans (KSPGs) within the corneal stroma. Increasing evidence indicates that lumican can serve as a regulatory molecule for several cellular processes including cell proliferation and migration. In the present study, we addressed the role of lumican in the process of PMN extravasation during the early inflammatory phase present in the healing of the corneal epithelium following debridement. We used Lum^-/- null mice and a novel transgenic mouse, Lum^-/-/Kera-Lum that expresses lumican only in the corneal stroma, to assess the role of lumican in PMN extravasation into injured corneas. Our results showed that PMNs did not readily invade injured corneas of Lum^-/- mice and this defect was rescued by the expression of lumican in the corneas of Lum^-/-/Kera-Lum mice. The presence of lumican in situ facilitates PMN infiltration into the peritoneal cavity by casein induced inflammation. Our findings are consistent with the notion that in addition to regulating the collagen fibril architecture, lumican acts to aid in neutrophil recruitment and invasion following corneal damage and inflammation. (Journal of Cell Sciences, in press)

3. Keratocan and Lumican Are Cleaved and Sustain CXCL1/KC in Corneal Inflammation

Keratocan and lumican are small leucine rich repeat keratan sulfate proteoglycans (KSPG) in the extracellular matrix (ECM) of the mammalian cornea. We examined the role of these proteoglycans in breakdown of the chemokine gradient and resolution of corneal inflammation. LPS injection induced 1) expression of the 52 kD keratocan core protein was diminished after 6 h, coincident with appearance of 34 kD and 37 kD bands; 2) presence of the 34/37kD doublet was dependent on neutrophil infiltration to the corneal stroma, as they were not detected in LPS injected corneas of chemokine receptor 2 (CXCR2) null mice, or in explanted corneas; 3) the 34/37kD doublet but not the 52kD core protein was detected in the anterior chamber, which drains the corneal stroma; and 4) CXCL1/KC was detected in the draining anterior chamber of LPS injected C57BL/6 mice, and was greatly elevated in keratocan null and in lumican null mice. Together, these findings indicate that the inflammatory response in the cornea is regulated by degradation of proteoglycan/CXCL1 complexes and diffusion to the anterior chamber, and is consistent with breakdown of a chemokine gradient and resolution of inflammation (Journal of Leukocytes Biology, in press).

4. Cell Therapy of Congenital Corneal Diseases with Umbilical Mesenchymal Stem Cells: Lumican Null Mice

Keratoplasty is the most effective treatment for corneal blindness, but suboptimal medical conditions and lack of qualified medical personnel and donated cornea often prevent the performance of corneal transplantation in developing countries. Our study aims to develop alternative treatment regimens for congenital corneal diseases of genetic mutation. Human mesenchymal stem cells isolated from neonatal umbilical cords were transplanted to treat thin and cloudy corneas of lumican null mice. Transplantation of umbilical mesenchymal stem cells significantly improved corneal transparency and increased stromal thickness of lumican null mice, but human umbilical hematopoietic stem cells failed to do the same. Further studies revealed that collagen lamellae were re-organized in corneal stroma of lumican null mice after mesenchymal stem cell transplantation. Transplanted umbilical mesenchymal stem cells survived in the mouse corneal stroma for more than 3 months with little or no graft rejection. In addition, these cells assumed a keratocyte phenotype, e.g., dendritic morphology, quiescence, expression of keratocyte unique keratan sulfated keratocan and lumican, and CD34. Moreover, umbilical mesenchymal stem cell transplantation improved host keratocyte functions, which was verified by enhanced expression of keratocan and aldehyde dehydrogenase class 3A1 in lumican null mice. Thus, umbilical mesenchymal stem cell transplantation is a promising treatment for congenital corneal diseases involving keratocyte dysfunction. (PLoS One, 5: 1-14, 2010)

5. Lumican Promotes Wound Healing by Enhancing Cell Proliferation via ERK Pathway in Cultured Human Corneal Epithelial Cell In Vitro

Lumican, a major Keratan sulfate proteoglycan in corneal stroma, plays an important role in corneal wound healing. Human telomerase immortalized corneal epithelial cells (HTCE) were used to examine the cellular mechanism by which lumican modulates wound healing. Administration of recombinant GST-lumican (glutathione S-tranferase-lumican fusion protein) isolated from E. coli transfected with pGEX-2T-Lum plasmid, greatly enhanced the closure of scratch wound with confluent HTCE cultures. GST-lumican stimulated cell proliferation as shown by a marked increase of Ki67 positive cells at the edge of scratch and away from wound site in comparison to that of control groups (without GST-Lum and GST only treatments). Activation of ERK and Smad3 via phosphorylation was observed in 10 min of adding GST-lumican, whereas phospho-p38MAPK was not detected within 1 hr after wounding in the presence or absence of GST-lumican. These findings suggest that GST-lumican stimulates cell migration and proliferation during wound healing in vitro. The lumican effects on wound healing might be mediated via the activation of ERK 1/2 and/or TGFβ signaling pathways.

6. Dysfunctional regulation of extracellular matrix assembly in a mouse model of human congenital stromal corneal dystrophy

Congenital stromal corneal dystrophy is associated with a mutant decorin gene, resulting in a truncated protein core lacking the C-terminal 33 amino acids. A mouse model of the human disease was developed to investigate the role(s) of decorin in matrix assembly in the normal development and pathobiology of the corneal stroma. Using a Cre-on approach, a transgenic mouse model was developed. Mutant decorin expression comparable to that in human congenital corneal stromal dystrophy was targeted to the cornea stroma. The transgenic cornea-specific decorin mutant mice developed cloudy corneas. Ultrastructural analysis demonstrated a comparable endothelium and Decemet’s membrane in transgenic and control mice. However, disorganized orthogonal lamellar structure was observed in the corneal stroma of the transgenic mice. This phenotype was most obvious in the posterior cornea. Lamellae with relatively normal collagen fibrils were separated by layers of abnormal filaments embedded in an abnormal lucent ground substance. Within these abnormal regions there was increased inter-filament/fibril spacing/organization. The abnormal regions were adjacent to keratocytes. Immunohistochemical analysis revealed reduced reactivity of both class II SLRPs, lumican and keratocan, in the posterior cornea in transgenic decorin mutant mice compared to wild type mice, suggesting an interaction between decorin and other SLRPs in the dysfunctional regulation. The phenotype in our mouse model was comparable to that in human disease of congenital stromal corneal dystrophy. Interaction of class II SLRRs may be involved in the dysfunctional regulation of corneal stroma matrix assembly caused by mutant decorin.

7. Constitutive Activation Of Notch Pathway Disrupts Eyelid Morphogenesis During And Following Mouse Development
In mammals, eyelids are of paramount importance for the formation of functional visual system, yet the cellular and molecular mechanisms underlying normal eyelid morphogenesis and homeostasis during and following development remain largely unknown. Notch signaling pathway plays a pivotal role in a wide variety of tissues morphogenesis and tumorigenesis. In the present study, we studied the effects of excessive Notch1 signaling activation in periocular mesenchymal cells of the cranial neural crest origin, which contribute to the formation of various ocular tissues, i.e., stromas of the eyelid, cornea, sclera, ciliary body and iris. Notch 1 intracellular domain (N1CD) was conditionally over-expressed in periocular mesenchymal cells of Kera-rtTA/tetO-Cre/Rosa^N1CD (KR/TC/Rosa^N1CD) triple transgenic mice treated with doxycycline (Dox) at different developmental stages, e.g., E14.5 (embryonic day 14.5) and post-natal day 2 (P2). These triple KR/TC/Rosa^N1CD mice exhibited variegation of eyelid anomalies depending on the age of initial Dox induction. When Dox was administered to embryos at E14.5, eyelid open at birth (EOB) phenotype was observed and animals suffered secondary exposure keratitis due to the malformation of tarsus and Meibomian glands. Interestingly, if Dox induction commenced at P2 at a time when eyelid closure had happened, the eyelid opening was much delayed and never reached full opening in these mice in comparison with the wild-type littermates. This phenotype resembles human congenital ptosis. Immunohistochemical analyses revealed that both Jagged and Notch1 were detected on the cytoplasmic membrane of the periocular mesenchymal cells of wild type mice, in contrast, Notch1 immunoreactivity was found mainly in the nuclei of these cells in the mutant mice. It is noteworthy that muscle differentiation marker genes, myoD, myogenein, and alpha-smooth actin expression were down-regulated. The observation implicates that constitutive Notch signaling may have perturbed myogenic differentiation of the periocular mesenchymal cells of neural crest origin, leading to the interruption of fetal eyelid closure during embryonic development and its postnatal re-opening. Thus, the KR/TC/Rosa^N1CD triple transgenic mouse is a novel model for examining the pathogenesis of EOB and congenital ptosis.
8. Corneal epithelial progenitors can maintain corneal surface homeostasis for a limited period of time following limbal deficiency

Basal cells of mouse corneal epithelial cells that derive from surface ectoderm remain undifferentiated (Krt12 negative). Corneal limbal epithelial debridement was performed with a Tet-On mouse model of Krt12^rt/wt/Tet-O-Cre/Rosa^mTmG/wt mice in which corneal epithelial cells normally expressed tomato-red fluorescence and became green due to the excision of LoxP-flanked tomato red fluorescence protein by Cre-recombinase upon Dox-induction. After limbal epithelial debridement, the healing of limbal epithelial deficiency was examined with a ZEISS fluorescence stereomicroscope. Corneal epithelium of adult Krt12^rt/wt/Tet-O-Cre/Rosa^mTmG/wt showed spiral green fluorescence upon Dox induction. Following entire corneal limbal epithelium debridement, central K12-positive green corneal epithelial cells migrated outwards and resurface the denuded limbus. Under continued Dox induction, the spiral EGFP pattern became disorganized. Cessation of Dox induction, green fluorescence vanished in 2 weeks; and re-administration of Dox lead to the formation of a mosaic green fluorescence of corneal epithelium. The number of green epithelial cells decreased after 6 weeks. Immunostaining with anti-Krt12, Krt13, and Krt15 antibodies revealed that both Krt12-positive and Krt13-positive cells existed on corneal surface. In addition, PAS positive goblet cells were present on the corneal surface epithelium. Krt12-positive corneal epithelial cells can temporarily maintains corneal epithelium integrity, but fail to rescue limbal deficiency.

C. Significance

Corneal transplantation, i.e., penetrating and lamellar keratoplasty, is the most effective treatment of corneal blindness caused by congenital gene mutation and trauma. However, the availability of donor corneas suitable for keratoplasty is decreasing. Thus, there is an urgent need to develop alternative strategies to replace keratoplasty in treating corneal diseases. We have carried our preliminary studies to examine the feasibility of human umbilical mesenchymal stem cells transplantation in treating corneal blindness resulting from congenital gene mutation and trauma in animal models. Our results suggest that umbilical stem cell transplantation can be beneficial in treating certain congenital and acquired corneal diseases in lieu of corneal transplantation for the treatment of corneal blindness.

Lumican is a Matrikine that regulates multiple cellular functions, e.g., cell proliferation and migration of corneal epithelial cells during wound healing besides serving as a regulator of collagen fibrillogenesis. The suggestion is best illustrated by the observation in which MSC transplanted into stroma effectively improve the physiological parameters, i.e., corneal transparency and stromal thickness, of diseased corneas of Lum-/- mice. These lumican functions on cellular activities must be mediated via the binding of lumican to a cell surface receptor(s). We hypothesize that the aforementioned perspective lumican receptor(s) mediates the biological functions of lumican on cell proliferation/survival, migration, differentiation and/or gene expression. In addition, results of our recent studies demonstrate that lumican and keratocan can bind chemokines, i.e., CXCL1. Thus, it serves as a modulator of corneal inflammation.

D. Plans

Alkali burn and laceration of corneal injuries result in scar tissue formation and impair vision. In the coming year, we will further explore the feasibility of using umbilical mesenchymal stem cells in treating corneal wounds, e.g., alkali burn and laceration, which result in scar tissue formation and loss of vision. In another series of GST-Lum fusion protein will be isolated and purified.

The purified lumican recombinant protein will be used to isolate and characterize the lumican receptor(s) of HTCE and inflammatory cells, e.g., PMN, macrophages. Recombinant truncated lumican mutant proteins will be prepared and then similarly tested for their biological functions using HTCE cells, PMNs and macrophages. Thus, the peptide sequence(s) that has biological activities in modulating cell functions can be identified. It is hoped that these experiments will allow us to design appropriate oligo peptides that may be used as therapeutic agents to improve wound healing in patient, e.g., diabetes.

In further experiments, Kera-rtTA and Krt12-rtTA drive mice will be used to ablate genes of interest, e.g., Ext1, PPAR-gamma, etc. Thus, we can determine their roles in ocular surface tissue morphogenesis during development and maintenance of homeostasis in adult.

E. Presentations and Publications

1. Zhang Y, Call MK, Yeh L-K, Liu H, Wang I-J, Chu P-H, Taketo MM, Jester J, Kao WW, and Liu C-Y. β-Catenin gain-of-function per se induces hyperplasia but its loss-of –function prevents excess FGF-7-induced neoplasia in the mouse cornea. J. Cell Sci. 123: 1285-1294, 2010 PM:20332116

2. Kao WW, and Liu C-Y. Corneal morphogenesis during development and wound healing. J. J. Ophthalmol. 54: 200-210 2010 PM:19560524

3. Liu H, Zhang J, Liu C-Y, Wang I-J, Sieber M, Chang J, Jester JV and Kao WW. Cell therapy of corneal diseases with umbilical mesenchymal stem cells: Lumican null mice. PLoS One 5: (5):e10707, 2010 PM:20502663

4. Nien CJ, Massel S, Lin G, Liu H, Paugh JR, Liu C-Y, Kao WW-Y and Jester JV. The development of Meibomian glands in mice. Mol. Vis. 29: 1132-1140, 2010

Abstracts: 10 abstracts were presented at 2010 ARVO annual meeting

Invited speakers: at International Symposium in Yamaguchi 2010

Trilogy of the Cornea : The Past, Present and Future; Wakayama Medical University