Self-complementary AAV vector therapy for treating corneal cloudiness of mucopolysaccharidosis type VII (MPS VII).

PURPOSE: To design a novel efficacious scAAV-Gusb viral vector for treating Mucopolysaccharidosis Type VII (MPS VII) caused by a mutation in the ß-Glu gene (Gusb allele). METHODS: ß-Glu expression of single-stranded AAV-Gusb (ssAAV-Gusb) and self-complementary AAV (scAAV-Gusb) vectors are tested with cultured murine Gusb fibroblasts. The scAAV-Gusb vector was chosen in further studies to prolong the life span and treat corneal pathology of Gusb mice via intrahepatic injection of neonates and intrastromal injection in adults, respectively. Corneal pathology was studied using HRT2 in vivo confocal microscope and histochemistry in mice corneas. RESULTS: Both ssAAV-Gusb and scAAV-Gusb vectors expressed murine ß-Glu in cultured Gusb fibroblasts. The scAAV-Gusb vector had higher transduction efficiency than the ssAAV-Gusb vector. To prolong the life span of Gusb mice, neonates (3 days old) were administered with scAAV-Gusb virus via intrahepatic injection. The treatment improves the survival rate of Gusb mice, prolonging the median survival rate from 22.5 weeks (untreated) to 50 weeks (treated). Thereafter, we determined the efficacy of the scAAV-Gusb virus in ameliorating corneal cloudiness observed in aged Gusb mice. Both corneal cloudiness and stroma thickness decreased, and there was the presence of ß-Glu enzyme activity in the Gusb corneas receiving scAAV-Gusb virus associated with morphology change of amoeboid stromal cells in untreated to characteristic dendritic keratocytes morphology after 4-12 weeks of scAAV-Gusb virus injection. CONCLUSION: Intrahepatic injection of scAAV-Gusb is efficacious in prolonging the life span of Gusb mice, and intrastromal injection can ameliorate corneal phenotypes. Both strategies can be adapted for treating other MPS.

The gene therapy for corneal pathology with novel nonsense cystinosis mouse lines created by CRISPR Gene Editing.

PURPOSE: Cystinosis is an autosomal recessive lysosomal storage disease (LSDs) caused by mutations in the gene encoding cystinosin (CTNS) that leads to cystine crystal accumulation in the lysosome that compromises cellular functions resulting in tissue damage and organ failure, especially in kidneys and eyes. However, the underlying molecular mechanism of its pathogenesis remains elusive. Two novel mice lines created via CRISPR are used to examine the pathogenesis of cystinosis in the kidney and cornea and the treatment efficacy of corneal pathology using self-complimentary Adeno-associated viral (scAAV-CTNS) vector. METHODS: The CRISPR technique generated two novel cystinotic mouse lines, Ctnsis1 (an insertional mutation) and Ctnsis2 (a nonsense mutation). Immune histochemistry, renal functions test and HRT2 in vivo confocal microscopy were used to evaluate the age-related renal pathogenesis and treatment efficacy of the scAAV-CTNS virus in corneal pathology. RESULTS: Both mutations lead to the production of truncated Ctns proteins. Ctnsis1 and Ctnsis 2 mice exhibit the characteristic of cystinotic corneal crystal phenotype at four-week-old. Treatment with the scAAV-CTNS viral vector decreased the corneal crystals in the treated mice cornea. Ctnsis 1 show renal abnormalities manifested by increased urine volume, reduced urine osmolality, and the loss of response to Desmopressin (dDAVP) at 22-month-old but Ctnsis2 don't manifest renal pathology up to 2 years of age. CONCLUSIONS: Both Ctnsis1 and Ctnsis2 mice exhibit phenotypes resembling human intermediate nephropathic and ocular cystinosis, respectively. scAAV-CTNS viral vectors reduce the corneal cystine crystals and have a great potential as a therapeutic strategy for treating patients suffering from cystinosis.

Trends in using mesenchymal stromal/stem cells (MSCs) in treating corneal diseases.

Since their inception in the 1960s-70s, mesenchymal stem/stromal cells (MSCs) have gained interest because of their differentiation potential, anti-inflammatory effects, and immune-modulating properties. Both cell-based and cell-free MSC treatments show healing capacity in injured tissues. Cell-based treatment comprises MSCs and all secreted products, whereas cell-free treatments include only the secreted products. MSCs are therapeutically administered to many damaged organs owing to their efficacy. Specifically, the eye is a unique organ system to study the effects of MSCs, as treatment is easily applied and measured owing to its external location. The eye holds an immune-privileged status, wherein inflammation and immune responses are innately down-regulated. As excessive inflammation in the cornea often leads to fibrosis and irreversible corneal hazing, many studies have investigated the anti-inflammatory and immune-modulating capacities of MSCs. Decades of research suggest that MSCs modulate the immune response by secreting cytokines, growth factors, and extracellular matrix proteins that inhibit the infiltration of inflammatory cells following injury and promote a healing phenotype via M2 macrophage polarization. MSCs have also shown trans-differentiation potential into cornea-specific cell types during the wound healing process, such as corneal epithelial, stromal, or endothelial cells. This review discusses recent investigations of MSC treatment in the cornea, focusing on therapeutic efficacy, mechanisms, and future directions.

Transient expression of Wnt5a elicits ocular features of pseudoexfoliation syndrome in mice.

PURPOSE: Pseudoexfoliation (PEX) syndrome is an age-related systemic disease with ocular manifestations. The development of animal models is critical in order to elucidate the cause of the disease and to test potential treatment regimens. The purpose of this study is to report phenotypes found in mouse eyes injected with Adenovirus coding Wnt5a. Some of the phenotypes resemble those found in PEX patients while others are different. METHODS: Recombinant Adenovirus coding Wnt5a or green fluorescent protein (GFP) were injected into mouse eyes. Two months after the injection, eyes were examined for PEX phenotypes using slit lamp, fluorescence stereomicroscope, histological staining, immunostaining and transmission electron microscope. RESULT: Certain ocular features of PEX syndrome were found in mouse eyes injected with recombinant Adenovirus coding Wnt5a. These features include accumulation of exfoliation-like extracellular material on surfaces of anterior segment structures and its dispersion in the anterior chamber, saw-tooth appearance and disrupted basement membrane of the posterior iris pigment epithelium, iris stromal atrophy and disorganized ciliary zonules. Ultrastructure analysis of the exfoliation material revealed that the microfibril structure found in this model was different from those of PEX patients. CONCLUSION: These features, resembling signs of ocular PEX syndrome in patients, suggest that new information obtained from this study will be helpful for developing better mouse models for PEX syndrome.

Role of EGF receptor signaling on morphogenesis of eyelid and meibomian glands.

The epidermal growth factor receptor (EGFR) signaling has a pivotal role in the regulation of morphogenesis during development and maintenance of homeostasis in adult eyelid and its adnexa. Studies have demonstrated that during eyelid morphogenesis the EGFR signaling pathway is responsible for keratinocyte and mesenchymal cell proliferation and migration at the eyelid tip. For meibomian gland morphogenesis, EGFR signaling activation stimulates meibomian gland epithelial cell proliferation. EGFR signaling pathway functions through multiple downstream signals such as ERK, Rho/ROCK and integrin and is regulated by a variety of upstream signals including Adam17, GPR48 and FGFR signaling. Herein we review the literature that describe the role of EGFR and its related signaling pathways in eyelid and meibomian gland morphogenesis.

Lumican Peptides: Rational Design Targeting ALK5/TGFBRI.

Lumican, a small leucine rich proteoglycan (SLRP), is a component of extracellular matrix which also functions as a matrikine regulating multiple cell activities. In the cornea, lumican maintains corneal transparency by regulating collagen fibrillogenesis, promoting corneal epithelial wound healing, regulating gene expression and maintaining corneal homeostasis. We have recently shown that a peptide designed from the 13 C-terminal amino acids of lumican (LumC13) binds to ALK5/TGFBR1 (type1 receptor of TGFß) to promote wound healing. Herein we evaluate the mechanism by which this synthetic C-terminal amphiphilic peptide (LumC13), binds to ALK5. These studies clearly reveal that LumC13-ALK5 form a stable complex. In order to determine the minimal amino acids required for the formation of a stable lumican/ALK5 complex derivatives of LumC13 were designed and their binding to ALK5 investigated in silico. These LumC13 derivatives were tested both in vitro and in vivo to evaluate their ability to promote corneal epithelial cell migration and corneal wound healing, respectively. These validations add to the therapeutic value of LumC13 (Lumikine) and aid its clinical relevance of promoting the healing of corneal epithelium debridement. Moreover, our data validates the efficacy of our computational approach to design active peptides based on interactions of receptor and chemokine/ligand.

Cell Therapy of Corneal Diseases.

Mesenchymal stem cells isolated from connective tissues are pluripotent and differentiate into phenotypes of connective tissue cell lineages (osteoblasts, chondrocytes, and adipocytes) in vitro and in vivo. They have been used to treat mouse models of connective tissue disease such as lumican-null (Lum) and mucopolysaccharidosis (Gusb) mice. Mesenchymal stem cells have unique immunosuppressive properties allowing evasion of host rejection; thus, they are valuable tools for cell therapy of congenital and acquired diseases involving immune dysfunction of multiple tissues including ocular surface tissues (cornea). We previously showed that human umbilical mesenchymal stem cells (UMSCs) modulated host immune responses, enabling them to survive xenograft transplantation. In vitro, UMSCs modulated inflammatory cells by inhibiting adhesion and invasion, and inducing cell death. UMSCs also regulated M1/M2 macrophage polarization and induced T-regulatory cell maturation from naive intraperitoneal cavity lavage cells. UMSCs exposed to inflammatory cells synthesized a rich extracellular glycocalyx composed of hyaluronan (HA) bound to the heavy chains (HCs) of inter-alpha-trypsin inhibitor (HC-HA), which contains tumor necrosis factor-α-stimulated gene 6 (TSG6) that catalyzes the transfer of HCs to HA, versican, and pentraxin-3. Our in vivo and in vitro results showed that the glycocalyx regulated inflammatory cells, allowing UMSCs to survive host immune rejection. Administration of antibodies against glycocalyx constituents or digestion with hyaluronidase and chondroitinase ABC abolished the UMSCs' ability to modulate immune responses. Treatment with anti-CD44 antibodies also diminished modulation of M2 macrophages by UMSCs, indicating that cell surface CD44 is required for correct UMSC glycocalyx assembly to modulate inflammatory cells.

A Case of Solitary Nonvascularized Corneal Epithelial Dysplasia.

Background. Epithelial dysplasia is categorized as conjunctival/corneal intraepithelial neoplasia which is a precancerous lesion. The lesion is usually developed at the limbal region and grows towards central cornea in association with neovascularization into the lesion. Here, we report a case of isolated nonvascularized corneal epithelial dysplasia surrounded by normal corneal epithelium with immune histochemical finding of ocular surface tissues cytokeratins, for example, keratin 13 and keratin 12. Case Presentation. A 76-year-old man consulted us for visual disturbance with localized opacification of the corneal epithelium in his left eye. His visual acuity was 20/20 and 20/200 in his right and left eye, respectively. Slit lamp examination showed a whitish plaque-like lesion at the center of his left corneal epithelium. No vascular invasion to the lesion was found. The lesion was surgically removed and subjected to histopathological examination and diagnosed as epithelial dysplasia. Amyloidosis was excluded by direct fast scarlet 4BS (DFS) staining. Immunohistochemistry showed that the dysplastic epithelial cells express keratin 13 and vimentin, but not keratin 12, indicating that the neoplastic epithelial cells lacked corneal-type epithelium differentiation. Conclusions. The lesion was diagnosed as nonvascularized epithelial dysplasia of ocular surface. Etiology of the lesion is not known.

Extrinsic and Intrinsic Mechanisms by Which Mesenchymal Stem Cells Suppress the Immune System.

Mesenchymal stem cells (MSCs) are a group of fibroblast-like multipotent mesenchymal stromal cells that have the ability to differentiate into osteoblasts, adipocytes, and chondrocytes. Recent studies have demonstrated that MSCs possess a unique ability to exert suppressive and regulatory effects on both adaptive and innate immunity in an autologous and allogeneic manner. A vital step in stem cell transplantation is overcoming the potential graft-versus-host disease, which is a limiting factor to transplantation success. Given that MSCs attain powerful differentiation capabilities and also present immunosuppressive properties, which enable them to survive host immune rejection, MSCs are of great interest. Due to their ability to differentiate into different cell types and to suppress and modulate the immune system, MSCs are being developed for treating a plethora of diseases, including immune disorders. Moreover, in recent years, MSCs have been genetically engineered to treat and sometimes even cure some diseases, and the use of MSCs for cell therapy presents new perspectives for overcoming tissue rejection. In this review, we discuss the potential extrinsic and intrinsic mechanisms that underlie MSCs' unique ability to modulate inflammation, and both innate and adaptive immunity.

The role of proteoglycans in the reactive stroma on tumor growth and progression.

The stroma surrounding tumors can either restrict or promote tumor growth and progression, and both the cellular and non-cellular components of the stroma play an active role. The cellular components in the surrounding stroma include tumor-associated fibroblasts, host tissue cells and immune cells. The non-cellular components, which form the extracellular matrix (ECM) scaffold, include proteoglycans, collagen, proteinases, growth factors and cytokines. For tumorigenesis to occur it is necessary for tumor cells to modify the surrounding stroma. Tumor cells have mechanisms for achieving this, such as co-opting fibroblasts and modifying the ECM they produce, degrading the surrounding ECM and/or synthesizing a favorable ECM to support invasion. Proteoglycans are an important component of the ECM and play an active role in tumor growth and progression. The expression and glycosylation patterns of proteoglycans are altered in the stroma surrounding tumors and these molecules may support or restrict tumor growth and progression depending on the type and stage of tumor. In the present review we discuss the difference between the tumor promoting and restricting stromal reactions surrounding tumors and the role proteoglycans play.

Mesenchymal stem cells for treating ocular surface diseases.

Mesenchymal stem cells (MSC) have become a promising tool for cell therapy in regenerative medicine. They are readily available, demonstrate powerful differentiation capabilities and present immunosuppressive properties that aid them in surviving from host immune rejection for its great potential use in allograft. Currently clinical trials are underway using MSC, both culture-expanded allogeneic and autologous, for the treatment of a range of diseases not treatable by conventional therapies. A vast array of studies has dedicated towards the use of MSC for treating corneal diseases with very promising outcomes. MSC have successfully differentiated into keratocytes both in vitro and in vivo, and corneal epithelial cells in vitro, but it is uncertain if MSC can assume corneal epithelial cells in vivo. However, to date few studies have unequivocally established the efficacy of MSC for treating corneal endothelial defects. Currently, the diversity in protocols of the isolation and expansion of MSC are hindering to the assessment of cell treatment ability and the further development of treatment regimens. Therefore, future studies should develop international standards for MSC isolation and characterization. In this review, we discuss recent advances in MSC for treating ocular surface diseases.

ABCB5 is a limbal stem cell gene required for corneal development and repair.

Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs), and LSC deficiency is a major cause of blindness worldwide. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts, a gene allowing for prospective LSC enrichment has not been identified so far. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5) marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs in mice and p63α-positive LSCs in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency.

Serum response factor: positive and negative regulation of an epithelial gene expression network in the destrin mutant cornea.

Increased angiogenesis, inflammation, and proliferation are hallmarks of diseased tissues, and in vivo models of these disease phenotypes can provide insight into disease pathology. Dstn(corn1) mice, deficient for the actin depolymerizing factor destrin (DSTN), display an increase of serum response factor (SRF) that results in epithelial hyperproliferation, inflammation, and neovascularization in the cornea. Previous work demonstrated that conditional ablation of Srf from the corneal epithelium of Dstn(corn1) mice returns the cornea to a wild-type (WT) like state. This result implicated SRF as a major regulator of genes that contributes to abnormal phenotypes in Dstn(corn1) cornea. The purpose of this study is to identify gene networks that are affected by increased expression of Srf in the Dstn(corn1) cornea. Microarray analysis led to characterization of gene expression changes that occur when conditional knockout of Srf rescues mutant phenotypes in the cornea of Dstn(corn1) mice. Comparison of gene expression values from WT, Dstn(corn1) mutant, and Dstn(corn1) rescued cornea identified >400 differentially expressed genes that are downstream from SRF. Srf ablation had a significant effect on genes associated with epithelial cell-cell junctions and regulation of actin dynamics. The majority of genes affected by SRF are downregulated in the Dstn(corn1) mutant cornea, suggesting that increased SRF negatively affects transcription of SRF gene targets. ChIP-seq analysis on Dstn(corn1) mutant and WT tissue revealed that, despite being present in higher abundance, SRF binding is significantly decreased in the Dstn(corn1) mutant cornea. This study uses a unique model combining genetic and genomic approaches to identify genes that are regulated by SRF. These findings expand current understanding of the role of SRF in both normal and abnormal tissue homeostasis.

Dexamethasone induces cross-linked actin networks in trabecular meshwork cells through noncanonical wnt signaling.

PURPOSE: Dexamethasone (DEX) regulates aqueous humor outflow by inducing a reorganization of the cytoskeleton to form cross-linked actin networks (CLANs) in trabecular meshwork (TM) cells. Rho-associated protein kinase (ROCK) has been demonstrated to have an important role in this process, but the upstream components leading to its activation remain elusive. The purpose of the study is to demonstrate that noncanonical Wnt signaling mediates the DEX-induced CLAN formation in TM cells. METHODS: The TM cells were treated with 100 nM DEX in low serum medium for over 7 days. The medium was changed every 3 days. The cells were harvested and subjected to molecular analysis for the expression of Wnt ligands. Stress fiber structures were revealed by Phalloidin staining. Lentivirus-based shRNA against noncanonical Wnt receptor (Ror2) was used to determine the role of noncanonical Wnt signaling in DEX-induced CLAN formation. RESULTS: The DEX induced stress fiber rearrangement in TM cells. A noncanonical Wnt ligand (Wnt5a) was upregulated by DEX as demonstrated by Wnt ligand degenerate PCR, real-time quantitative PCR (qRT-PCR), and Western blotting. Knocking-down Ror2, the receptor of noncanonical Wnt signaling, abolished the effects of DEX on the TM cells. CONCLUSIONS: Our data suggest that DEX induces the upregulation of noncanonical Wnt ligand Wnt5a. Recombinant WNT5a protein induces CLAN formation through the noncanonical Wnt receptor ROR2/RhoA/ROCK signaling axis. Given the similarities between DEX-induced ocular hypertension and primary open-angle glaucoma, our results provide a mechanism of action for applying ROCK inhibitor to treat primary open-angle glaucoma.

Transplantation of human umbilical mesenchymal stem cells cures the corneal defects of mucopolysaccharidosis VII mice.

Mucopolysaccharidosis (MPS) are a family of related disorders caused by a mutation in one of the lysosomal exoglycosidases which leads to the accumulation of glycosaminoglycans (GAGs). MPS VII, caused by a mutation in ß-glucuronidase, manifests hepatomegaly, skeletal dysplasia, short stature, corneal clouding, and developmental delay. Current treatment regimens for MPS are not effective for treating corneal clouding and impaired mental development. We hypothesized that human umbilical mesenchymal stem cells (UMSCs) transplanted into the corneal stroma could participate in the catabolism of GAGs providing a means of cell therapy for MPS. For such treatment, human UMSCs were intrastromally transplanted into corneas of MPS VII mice. UMSC transplantation restored the dendritic and hexagonal morphology of host keratocytes and endothelial cells, respectively, and in vivo confocal microscopy (HRT-II) revealed reduced corneal haze. Immunohistochemistry using antibodies against heparan sulfate and chondroitin sulfate chains as well as lysosomal-associated membrane protein 2 revealed a decrease in GAG content and both lysosomal number and size in the treated corneas. Labeling UMSC intracellular compartments prior to transplantation revealed the distribution of UMSC vesicles throughout the corneal stroma and endothelium. An in vitro coculture assay between skin fibroblasts isolated from MPS VII mice and UMSC demonstrated that neutral vesicles released by the UMSC are taken up by the fibroblasts and proceed to fuse with the acidic lysosomes. Therefore, transplanted UMSCs participate both in extracellular GAG turnover and enable host keratocytes to catabolize accumulated GAG products, suggesting that UMSC could be a novel alternative for treating corneal defects associated with MPS and other congenital metabolic disorders.

Lumican expression, localization and antitumor activity in prostate cancer.

The stromal reaction surrounding tumors leads to the formation of a tumor-specific microenvironment, which may play either a restrictive role or a supportive role in the growth and progression of the tumors. Lumican, a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM), regulates collagen fibrillogenesis. Recently, lumican has also been shown to regulate cell behavior during embryonic development, tissue repair and tumor progression. The role of lumican in cancer varies according to the type of tumor. In this study we analyze the role of lumican in the pathogenesis of prostate cancer both in vivo and in vitro. Overall lumican up-regulation was observed in the primary tumors analyzed through both real-time PCR and immunostaining. The increase in lumican expression was observed in the reactive stroma surrounding prostate primary tumors with fibrotic deposition surrounding the acinar glands. In vitro analysis demonstrated that lumican inhibited both the migration and invasion of metastatic prostate cancer cells isolated from lymph node, bone and brain. Moreover, prostate cancer cells seeded on lumican presented a decrease in the formation of cellular projections, lamellipodia detected by a decreased rearrangement in ZO-1, keratin 8/18, integrin ß1 and MT1-MMP, and invadopodia detected by disruption of α-smooth muscle actin, cortactin and N-WASP. Moreover, a significant increase in prostate cancer cell invasion was observed through the peritoneum of lumican knockout mice, further demonstrating the restrictive role lumican present in the ECM has on prostate cancer invasion. In conclusion, lumican present in the reactive stroma surrounding prostate primary tumors plays a restrictive role on cancer progression, and we therefore postulate that lumican could be a valuable marker in prostate cancer staging.

Mastermind-like transcriptional co-activator-mediated Notch signaling is indispensable for maintaining conjunctival epithelial identity.

Conjunctival goblet cells primarily synthesize mucins to lubricate the ocular surface, which is essential for normal vision. Notch signaling has been known to associate with goblet cell differentiation in intestinal and respiratory tracts, but its function in ocular surface has yet to be fully characterized. Herein, we demonstrate that conditional inhibition of canonical Notch signaling by expressing dominant negative mastermind-like 1 (dnMaml1) in ocular surface epithelia resulted in complete suppression of goblet cell differentiation during and subsequent to development. When compared with the ocular surface of wild-type mice (OS(Wt)), expression of dnMaml1 at the ocular surface (OS(dnMaml1)) caused conjunctival epithelial hyperplasia, aberrant desquamation, failure of Mucin 5ac (Muc5ac) synthesis, subconjunctival inflammation and epidermal metaplasia in cornea. In addition, conditional deletion of Notch1 from the ocular surface epithelia partially recapitulated OS(dnMaml1) phenotypes. We have demonstrated that N1-ICD (Notch1 intracellular domain) transactivated the mouse Krüppel-like factor 4 (Klf) promoter and that Klf4 directly bound to and significantly potentiated the Muc5ac promoter. By contrast, OS(dnMaml1) dampened Klf4 and Klf5 expression, and diminished Muc5ac synthesis. Collectively, these findings indicated that Maml-mediated Notch signaling plays a pivotal role in the initiation and maintenance of goblet cell differentiation for normal ocular surface morphogenesis and homeostasis through regulation of Klf4 and Klf5.

Bone marrow mesenchymal stem cells can differentiate and assume corneal keratocyte phenotype.

It remains elusive as to what bone marrow (BM) cell types infiltrate into injured and/or diseased tissues and subsequently differentiate to assume the phenotype of residential cells, for example, neurons, cardiac myocytes, keratocytes, etc., to repair damaged tissue. Here, we examined the possibility of whether BM cell invasion via circulation into uninjured and injured corneas could assume a keratocyte phenotype, using chimeric mice generated by transplantation of enhanced green fluorescent protein (EGFP)(+) BM cells into keratocan null (Kera(-/-)) and lumican null (Lum(-/-)) mice. EGFP(+) BM cells assumed dendritic cell morphology, but failed to synthesize corneal-specific keratan sulfate proteoglycans, that is KS-lumican and KS-keratocan. In contrast, some EGFP(+) BM cells introduced by intrastromal transplantation assumed keratocyte phenotypes. Furthermore, BM cells were isolated from Kera-Cre/ZEG mice, a double transgenic mouse line in which cells expressing keratocan become EGFP(+) due to the synthesis of Cre driven by keratocan promoter. Three days after corneal and conjunctival transplantations of such BM cells into Kera(-/-) mice, green keratocan positive cells were found in the cornea, but not in conjunctiva. It is worthy to note that transplanted BM cells were rejected in 4 weeks. MSC isolated from BM were used to examine if BM mesenchymal stem cells (BM-MSC) could assume keratocyte phenotype. When BM-MSC were intrastromal-transplanted into Kera(-/-) mice, they survived in the cornea without any immune and inflammatory responses and expressed keratocan in Kera(-/-) mice. These observations suggest that corneal intrastromal transplantation of BM-MSC may be an effective treatment regimen for corneal diseases involving dysfunction of keratocytes.

TRPV1 involvement in inflammatory tissue fibrosis in mice.

We examined whether absence or blocking of transient receptor potential vanilloid subtype 1 (TRPV1) affects the level of inflammation and fibrosis/scarring during healing of injured tissue using an alkali burn model of cornea in mice. A cornea burn was produced with 1 N NaOH instilled into one eye of TRPV1-/- (KO) (n = 88) or TRPV1+/+ (n = 94) mice. Examinations of the corneal surface and eye globe size suggested that the loss of TRPV1 suppressed inflammation and fibrosis/scarring after alkali burn, and this was confirmed by histology, IHC, and gene expression analysis. The loss of TRPV1 inhibited inflammatory cell invasion and myofibroblast generation in association with reduction of expression of proinflammatory and profibrogenic components. Experiments of bone marrow transplantation between either genotype of mice showed that KO corneal tissue resident cells, but not KO bone marrow-derived cells, are responsible for KO-type wound healing with reduced inflammation and fibrosis. The absence of TRPV1 attenuated expression of transforming growth factor ß 1 (TGFß1) and other proinflammatory gene expression in cultured ocular fibroblasts, but did not affect TGFß1 expression in macrophages. Loss of TRPV1 inhibited myofibroblast transdifferentiation in cultured fibroblasts. Systemic TRPV1 antagonists reproduced the KO type of healing. In conclusion, absence or blocking of TRPV1 suppressed inflammation and fibrosis/scarring during healing of alkali-burned mouse cornea. TRPV1 is a potential drug target for improving the outcome of inflammatory/fibrogenic wound healing.

Changes in corneal basal epithelial phenotypes in an altered basement membrane.

BACKGROUND: To examine the corneal epithelial phenotype in an altered basement membrane. METHODOLOGY/PRINCIPAL FINDINGS: Corneas from 9 patients with symptoms of continuous unstable corneal curvature (CUCC) were harvested by penetrating keratoplasty and subjected to histology examination and immunohistochemical staining with transactivating and N-terminally truncated pP63 transcript (ΔNp63), cytokeratin 3 (Krt3), ATP-binding cassette sub-family G member 2 (ABCG2), connexin 43 (CX43), p38 mitogen-activated protein kinases (p38MAPK), activating protein 2 (TFAP2), and extracellular signal-regulated kinase (Erk1/2) monoclonal antibodies. Positive immunostaining with ABCG2, p38MAPK, and TFAP2 monoclonal antibodies was observed in the basal epithelial cells of CUCC patients, and CX43 and ΔNp63 were detected in the full-thickness epithelial cells of CUCC patients. CONCLUSIONS/SIGNIFICANCE: Our results indicate that alteration of the corneal basement membrane induces a de-differentiation-like phenotype in corneal basal epithelial cells.