UNIVERSITY of CINCINNATI
DEPARTMENT OF OPHTHALMOLOGY

by: Winston  Kao, Ph.D.

PROGRESS REPORT

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. We have demonstrated that UMSC transplantation can cure cloudy and thin corneas of Lum knockout mice and allow the regeneration of transparent cornea and endothelium following alkali-burn. 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. These lumican functions on cellular activities are mediated via the binding of lumican to a cell surface receptor(s). In addition, results of our recent studies demonstrate that lumican and keratocan can bind chemokines, i.e., CXCL 1. Thus, it serves as a modulator of corneal inflammation.

Statement We have made Significant progress in developing the technology of transplantation of human umbilical mesenchymal stem cells in curing congenital and acquired corneal diseases, such as lumican knockout mice, limbal deficiency and alkali burns, respectively. Our preliminary results indicate that intrastromal MSC transplantation prevents corneal cloudiness and ulceration caused by alkali burn. It is worthy to note when MSC were transplanted into the anterior chamber they differentiated to assume corneal endothelial cells phenotype. The MSC transplantation also allows the regeneration of corneal epithelial stem/progenitor cells of limbal stem cells deficiency (LSCD). We have demonstrated allograft of cultured hair follicle stem cells were able to cure LSCD. In attempt to examine the molecular and cellular mechanism of dry eye, a disease affecting millions of Americans, 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. The preliminary studies with mesenchymal stem cells lead to a grant application submitted to NIH/NEI (pending funding)

From hair to cornea: therapeutic use of hair follicle-derived stem cells in the treatment of limbal stem cell deficiency In this study we demonstrated that hair follicle stem cells could be expanded in culture and grafted to cure LSCD in a transgeniC mouse model, Krt1ztTAlrfTAltetO_creIROSAmTmG. The HFSC transplant was able to reconstruct the ocular surface in 80% of the transplanted animals; differentiating into cells with a corneal epithelial phenotype, expressing Krt12, repopulating the corneal SC pool while suppressing vascularization and conjunctival in-growth. These data highlight the therapeutic properties of using HFSC to treat LSCD in a mouse model while demonstrating a strong translational potential and points to the niche as a key factor for determining stem cell differentiation.

De-differentiation of corneal basal epithelial cells in an altered basement membrane We examined the role of basement membrane on corneal epithelial phenotype in human ocular surface disorder with symptom of continuous unstable corneal curvature (CUCC). Corneas of CUCC patients were harvested by penetrating keratoplasty and subjected to histology examination and immunohistochemical staining with markers of corneal epithelial stem/progenitor cells, and corneal-type epithelial differentiation. Positive immunostaining with ABCG2, p38MAPK, and AP2 monoclonal antibodies could be found in the basal epithelial cells of CUCC, and CX43 and flNp63 were detected in the full thickness epithelial cells of CUCC. Our results indicated that the alteration of corneal basement membrane will result in a dedifferentiated status of corneal basal epithelial cells.

Cell Therapy of Congenital Corneal Diseases with Umbilical Mesenchymal Stem Cells: Lumican Null Mice Our study aims to develop alternative treatment regimens for congenital corneal diseases of genetiC mutation. Transplantation of umbilical mesenchymal stem cells significantly improved corneal transparency and increased stromal thickness of lumican null mice by irnproving the collagen lamellae, but human umbilical hematopoietic stem cells failed to do the same. 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 3A 1 in lumican null mice. Umbilical mesenchymal stem cell transplantation is a promising treatment for congenital corneal diseases involving keratocyte dysfunction. Unlike donated corneas, umbilical mesenchymal stem cells are easily isolated, expanded, stored, and can be quickly recovered from liquid nitrogen when a patient is in urgent need.

Lumican is required for neutrophil extravasation following corneal injury and 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. In the study, we showed that lumican had a role in extravasation of polymorph nuclear leukocytes (PMNs) during the early inflammatory phase present in the healing of the corneal epithelium following debridement using Lum-I-mice and a novel bi-transgenic mouse, Lum-l-lKera-Lum. Our results showed that PMNs did not readily invade injured corneas of Lum-I-mice and this defect was rescued by the expression of lumican in the corneas of Lum-1-lKera-Lum mice. The presence of lumican in situ facilitates PMN infiltration into the peritoneal cavity in casein-induced inflammation. Our findings are consistent with the notion that in addition to regulating the collagen ‘fibril architecture, lumican acts to aid neutrophil recruitment and invasion following corneal damage and inflammation.

Regulation of corneal inflammation by neutrophil-dependent cleavage of keratan sulfate proteoglycans as a model for breakdown of the chemokine gradient We examined the role of lumican and keratocan in the breakdown of the chemokine gradient and resolution of corneal inflammation. LPS was injected into the corneal stroma of C57BU6 mice, and corneal extracts were examined by immunoblot analysis. We found reduced expression of the 52-kD keratocan protein after 6 h and conversely, increased expression of 34/37 kD immunoreactive products. Further, appearance of the 34/37-kD proteins was dependent on neutrophil infiltration to the cornea, as the appearance of these products was coincident with neutrophil infiltration, and the 34/37-kD products were not detected in explanted corneas or in CXCR2-1-corneas with deficient neutrophil recruitment. Furthermore, the 34/37-kD products and CXCL 1/KC were detected in the anterior chamber, into which the corneal stroma drains; and CXCL 1/KC was elevated significantly in Kera-I-and Lum-I-mice. Together, these findings indicate that the inflammatory response in the cornea is regulated by proteoglycan/CXCL 1 complexes, and their diffusion into the anterior chamber is consistent with release of a chemokine gradient and resolution of inflammation.

Knockdown of Zebrafish Lumican Gene (zlum) Causes Scleral Thinning and Increased Size of Scleral Coats 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. Like human (hLUM) and mouse (mLum), zLum consists of three exons, two introns, and a TATA box-less promoter at the 5-flanking region of the transcription initiation site. zLum encodes 344 amino acids. zLum shares 51 % amino acid sequence identity with human lumican. Similar to hLUM and mLum, zLum mRNA is expressed in the eye and many other tissues, such as brain, muscle, and liver as well. Transgenic zebrafish harboring an enhanced GFP reporter gene construct downstream of a 1.7-kb zLum 5′-flanking region displayed enhanced GFP expression in the cornea and sclera, as well as throughout the body. Down regulation of zLum expression by antisense zLum morpholinos manifested ocular enlargement resembling 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 and pirenzepine, effectively subdued the ocular enlargement caused by morpholinos in in vivo zebrafish larvae assays. The observation suggests that zebrafish can be used as an in vivo model for screening compounds in treating myopia.

Monoallelic Expression of Krt12 Gene during Cornealtype Epithelium Differentiation of Limbal Stem Cells We characterized a Krt12-Cre knock-in mouse line for corneal epithelium-specific gene ablation and to analyze the allelic selection of the keratin 12 (Krt12) gene during corneal type-epithelium differentiation. The expression patterns of several re£orter genes in the corneas of bitransgenic Krt12Crel+IROSAEGFP, Krt12CreI+IZEG, and Krt12Cre +IZAP mouse lines were examined. Krt12 and cre recombinase (Cre) immunostaining was performed. Corneal epithelial cells from bitransgenic Krt12crel+ IROSAEGFP mice were examined by fluorescence-activated cell sorter. We found that mosaic and spiral expression patterns of EGFP were observed in young and adult bitransgenic Krt12creI+IZEG mice, respectively. Immunostaining revealed that Cre-cells were also Krt12 negative in the corneal epithelia of Krt12Crel-1ZAP mice. Using FACS analysis, 60% to 70% of the corneal epithelial cells from Krt12Crel+IROSAEGFP mice were EGFP positive, whereas 20% to 30% were negative. RTPCR revealed that EGFP+ cells express both Krt12Cre and Krt12+ alleles, whereas EGFP-cells express only Krt12+ . In the Krt12Crel+cornea, the number of epithelial cells expressing Cre is the same as that found in Krt12Crelcre, which can be explained by the fragility of corneal epithelial cells that did not produce Krt12 because the Krt12Cre allele was not transcribed. These observations are consistent with the notion that clonal limbal stem cells randomly activate Krt12 alleles in the process of terminal differentiation. The authors suggest that this selection is advantageous for retaining epithelial cells expressing the Krt12+ allele and that it allows tolerance to structural mutations of Krt12.

Notch gain of function in mouse periocular mesenchyme down regulates FoxL2 and impairs eyelid levator muscle formation, leading to congenital blepharophimosis notch signaling is pivotal for the morphogenesis and homeostasis of many tissues. We found that aberrant Notch activation in mouse neural-crest-derived periocular mesenchymal cells (POMCs), which contribute to the formation of corneal and eyelid stroma, results in blepharophimosis. Compound transgenic mice over expressing the Notch1 intracellular domain (N1-ICD) in POMCs (POMCN1-ICD) showed relatively minor effects on the cornea, but increased cell apoptosis and decreased cell proliferation during eyelid morphogenesis. Eyelid closure at E 15.5 and eyelid formation at birth were incomplete. In further analyses, overexpression of N 1-ICD impaired eyelid levator smooth (MOeller) muscle formation by down regulating the transcription factor FoxL2. This is similar to the effect of haploinsufficiency of FOXL2 in humans, which results in type II BPES (blepharophimosis, ptosis and epicanthus inversus syndrome). In vitro studies showed that FoxL2 expression is augmented by a low dose of N1-ICD but was down regulated by a high dose, depending on the extent of Hes-1 and Hey-1 activation. Moreover, transfection of CMV-FoxL2 enhanced a-SMA promoter activity. These data strongly imply that a physiologically low level of Notch1 is crucial for proper FoxL2 expression in POMCs, which is, in turn, essential for MOeller muscle formation and normal eyelid development.

Lumican binds TGF-J3 type I receptor and stimulates wound healing of culture corneal epithelia cells Lumican, a member of Small Leucine-rich Proteoglycan family, plays an important role in corneal wound healing. We reported that lumican binds TGFJ3 type I receptor (TBR1) by two-hybrid analysis. To elucidate the biological function of lumican, we examined the signaling of lumican administered to human telomerase immortalized corneal epithelial cells (HTCE) cells and 293 cells. Recombinant GST-Iumican (glutathione Stranferase-Iumican fusion protein) was isolated from E. coli transfected with pGEX-2T-Lum plasmid, and subsequently purified using glutathione-Sepharose affinity column. Closure of scratch wound with confluent HTCE cultures was used to examine the wound healing process in the presence or absence of recombinant GST -Iumican, in vitro. In the HTCE scratch wound model, expression of TBR 1 and activation of Smad2 and Smad3 via phosphorylation were observed in 5 min of adding GST-Iumican and in 30 min phosphorylation of ERK1/2 was observed. Phospho-p38MAPK expression was not detected up to 1 hr in the presence or absence of GST-Iumican. Co-localization of GST-Iumican with Tbr1 was observed diffusely in 293 cells after 4°C incubation, at 15 min further 37°C incubation limited the immunolocalization of GST-Iumican and TBR1 to around the nuclei not whole cytoplasm. At 30 min, immunoreactivity of them was not observed. The results were consistent with the hypothesis that lumican binds to Tbr1 to exert its effect on cell proliferation and healing of scratched wound of HTCE cells.