Heterozygous Loss of Yap1 in Mice Causes Progressive Cataracts.

Purpose: Yap1 encodes an evolutionarily conserved transcriptional coactivator and functions as a down-stream effector of the Hippo signaling pathway that controls tissue size and cell growth. Yap1 contributes to lens epithelial development. However, the effect of Yap1 haplodeficiency on the lens epithelium and its role in the development of cataracts has not been reported. The aim of the current study is to investigate Yap1 function and its regulatory mechanisms in lens epithelial cells (LECs). Methods: Lens phenotypes were investigated in Yap1 heterozygous mutant mice by visual observation and histological and biochemical methods. Primary LEC cultures were used to study regulatory molecular mechanism. Results: The heterozygous inactivation of Yap1 in mice caused cataracts during adulthood with defective LEC phenotypes. Despite a normal early development of the eye including the lens, the majority of Yap1 heterozygotes developed cataracts in the first six months of age. Cataract was preceded by multiple morphological defects in the lens epithelium, including decreased cell density and abnormal cell junctions. The low LEC density was coincident with reduced LEC proliferation. In addition, expression of the Yap1 target gene Crim1 was reduced in the Yap1+/- LEC, and overexpression of Crim1 restored Yap1+/- LEC cell proliferation in vitro. Conclusions: Homozygosity of the Yap1 gene was critical for adequate Crim1 expression needed to maintain the constant proliferation of LEC and to maintain a normal-sized lens. Yap1 haplodeficiency leads to cataracts.

miR-15a/16 reduces retinal leukostasis through decreased pro-inflammatory signaling.

BACKGROUND: Hyperglycemia is a significant risk factor for diabetic retinopathy and induces increased inflammatory responses and retinal leukostasis, as well as vascular damage. Although there is an increasing amount of evidence that miRNA may be involved in the regulation in the pathology of diabetic retinopathy, the mechanisms by which miRNA mediate cellular responses to control onset and progression of diabetic retinopathy are still unclear. The purpose of our study was to investigate the hypothesis that miR-15a/16 inhibit pro-inflammatory signaling to reduce retinal leukostasis. METHODS: We generated conditional knockout mice in which miR-15a/16 are eliminated in vascular endothelial cells. For the in vitro work, human retinal endothelial cells (REC) were cultured in normal (5 mM) glucose or transferred to high glucose medium (25 mM) for 3 days. Transfection was performed on REC in high glucose with miRNA mimic (hsa-miR-15a-5p, hsa-miR-16-5p). Statistical analyses were done using unpaired Student t test with two-tailed p value. p < 0.05 was considered significant. Data are presented as mean ± SEM. RESULTS: We demonstrated that high glucose conditions decreased expression of miR-15a/16 in cultured REC. Overexpression of miR-15a/16 with the mimic significantly decreased pro-inflammatory signaling of IL-1ß, TNFα, and NF-κB in REC. In vivo data demonstrated that the loss of miR-15a/16 in vascular cells led to increased retinal leukostasis and CD45 levels, together with upregulated levels of IL-1ß, TNFα, and NF-κB. CONCLUSIONS: The data indicate that miR-15a/16 play significant roles in reducing retinal leukostasis, potentially through inhibition of inflammatory cellular signaling. Therefore, we suggest that miR-15a/16 offer a novel potential target for the inhibition of inflammatory mediators in diabetic retinopathy.

Loss of Neurokinin-1 Receptor Alters Ocular Surface Homeostasis and Promotes an Early Development of Herpes Stromal Keratitis.

Substance P neuropeptide and its receptor, neurokinin-1 receptor (NK1R), are reported to present on the ocular surface. In this study, mice lacking functional NK1R exhibited an excessive desquamation of apical corneal epithelial cells in association with an increased epithelial cell proliferation and increased epithelial cell density, but decreased epithelial cell size. The lack of NK1R also resulted in decreased density of corneal nerves, corneal epithelial dendritic cells (DCs), and a reduced volume of basal tears. Interestingly, massive accumulation of CD11c+CD11b+ conventional DCs was noted in the bulbar conjunctiva and near the limbal area of corneas from NK1R-/- mice. After ocular HSV-1 infection, the number of conventional DCs and neutrophils infiltrating the infected corneas was significantly higher in NK1R-/- than C57BL/6J mice. This was associated with an increased viral load in infected corneas of NK1R-/- mice. As a result, the number of IFN-γ-secreting virus-specific CD4 T cells in the draining lymph nodes of NK1R-/- mice was much higher than in infected C57BL/6J mice. An increased number of CD4 T cells and mature neutrophils (CD11b+Ly6ghigh) in the inflamed corneas of NK1R-/- mice was associated with an early development of severe herpes stromal keratitis. Collectively, our results show that the altered corneal biology of uninfected NK1R-/- mice along with an enhanced immunological response after ocular HSV-1 infection causes an early development of herpes stromal keratitis in NK1R-/- mice.

Study of corneal epithelial progenitor origin and the Yap1 requirement using keratin 12 lineage tracing transgenic mice.

Key issues in corneal epithelium biology are the mechanism for corneal epithelium stem cells to maintain the corneal epithelial homeostasis and wound healing responses, and what are the regulatory molecular pathways involved. There are apparent discrepancies about the locations of the progenitor populations responsible for corneal epithelial self-renewal. We have developed a genetic mouse model to trace the corneal epithelial progenitor lineages during adult corneal epithelial homeostasis and wound healing response. Our data revealed that the early corneal epithelial progenitor cells expressing keratin-12 originated from limbus, and gave rise to the transit amplifying cells that migrated centripetally to differentiate into corneal epithelial cells. Our results support a model that both corneal epithelial homeostasis and wound healing are mainly maintained by the activated limbal stem cells originating form limbus, but not from the corneal basal epithelial layer. In the present study, we further demonstrated the nuclear expression of transcriptional coactivator YAP1 in the limbal and corneal basal epithelial cells and its essential role for maintaining the high proliferative potential of those corneal epithelial progenitor cells in vivo.

IKK2 inhibition using TPCA-1-loaded PLGA microparticles attenuates laser-induced choroidal neovascularization and macrophage recruitment.

The inhibition of NF-κB by genetic deletion or pharmacological inhibition of IKK2 significantly reduces laser-induced choroid neovascularization (CNV). To achieve a sustained and controlled intraocular release of a selective and potent IKK2 inhibitor, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1) (MW: 279.29), we developed a biodegradable poly-lactide-co-glycolide (PLGA) polymer-delivery system to further investigate the anti-neovascularization effects of IKK2 inhibition and in vivo biosafety using laser-induced CNV mouse model. The solvent-evaporation method produced spherical TPCA-1-loaded PLGA microparticles characterized with a mean diameter of 2.4 »m and loading efficiency of 80%. Retrobulbar administration of the TPCA-1-loaded PLGA microparticles maintained a sustained drug level in the retina during the study period. No detectable TPCA-1 level was observed in the untreated contralateral eye. The anti-CNV effect of retrobulbarly administrated TPCA-1-loaded PLGA microparticles was assessed by retinal fluorescein leakage and isolectin staining methods, showing significantly reduced CNV development on day 7 after laser injury. Macrophage infiltration into the laser lesion was attenuated as assayed by choroid/RPE flat-mount staining with anti-F4/80 antibody. Consistently, laser induced expressions of Vegfa and Ccl2 were inhibited by the TPCA-1-loaded PLGA treatment. This TPCA-1 delivery system did not cause any noticeable cellular or functional toxicity to the treated eyes as evaluated by histology and optokinetic reflex (OKR) tests; and no systemic toxicity was observed. We conclude that retrobulbar injection of the small-molecule IKK2 inhibitor TPCA-1, delivered by biodegradable PLGA microparticles, can achieve a sustained and controllable drug release into choroid/retina and attenuate laser-induced CNV development without causing apparent systemic toxicity. Our results suggest a potential clinical application of TPCA-1 delivered by microparticles in treatment of CNV in the patients with age-related macular degeneration and other retinal neovascularization diseases.

IL-2/anti-IL-2 antibody complex treatment inhibits the development but not the progression of herpetic stromal keratitis.

The IL-2/anti-IL-2 Ab immunocomplex has recently been shown to expand the naturally occurring pool of CD4(+)Foxp3(+) regulatory T cells (Tregs). In this study, we show that administration of the IL-2/anti-IL-2 Ab immunocomplex to C57BL/6 mice, prior to corneal HSV-1 infection, significantly increased the pool of Foxp3(+) Tregs when measured at early time points postinfection. Increased numbers of Foxp3(+) Tregs on days 2 and 4 postinfection resulted in a marked reduction in the development of severe herpetic stromal keratitis (HSK). When compared with corneas from the control group, corneas from the immunocomplex-treated group showed a significant reduction in the amount of infectious virus on day 2 but not on day 4 postinfection. Reduced viral load was associated with a 2-fold increase in NK cell numbers in corneas from the immunocomplex-treated group of mice. Moreover, a dramatic reduction in the influx of CD4 T cells in inflamed corneas was determined on days 7 and 16 postinfection in the immunocomplex-treated group of infected mice. Immunocomplex treatment given on days 5, 6, and 7 postinfection significantly increased Foxp3(+) Tregs in draining lymph nodes and in the spleen but failed to reduce the severity of HSK. In terms of the influx of CD4 T cells and granulocytes into inflamed corneas, no significant differences were noted between both groups of mice on day 16 postinfection. Our findings demonstrate that increasing Foxp3(+) Tregs early but not late postinfection in secondary lymphoid tissues is more efficacious in controlling the severity of HSK.

IKK2 inhibition attenuates laser-induced choroidal neovascularization.

Choroidal neovascularization (CNV) is aberrant angiogenesis associated with exudative age-related macular degeneration (AMD), a leading cause of blindness in the elderly. Inflammation has been suggested as a risk factor for AMD. The IKK2/NF-κB pathway plays a key role in the inflammatory response through regulation of the transcription of cytokines, chemokines, growth factors and angiogenic factors. We investigated the functional role of IKK2 in development of the laser-induced CNV using either Ikk2 conditional knockout mice or an IKK2 inhibitor. The retinal neuronal tissue and RPE deletion of IKK2 was generated by breeding Ikk2(-/flox) mice with Nestin-Cre mice. Deletion of Ikk2 in the retina caused no obvious defect in retinal development or function, but resulted in a significant reduction in laser-induced CNV. In addition, intravitreal or retrobulbar injection of an IKK2 specific chemical inhibitor, TPCA-1, also showed similar inhibition of CNV. Furthermore, in vitro inhibition of IKK2 in ARPE-19 cells significantly reduced heat shock-induced expression of NFKBIA, IL1B, CCL2, VEGFA, PDGFA, HIF1A, and MMP-2, suggesting that IKK2 may regulate multiple molecular pathways involved in laser-induced CNV. The in vivo laser-induced expression of VEGFA, and HIF1A in RPE and choroidal tissue was also blocked by TPCA-1 treatment. Thus, IKK2/NF-κB signaling appears responsible for production of pro-inflammatory and pro-angiogenic factors in laser-induced CNV, suggesting that this intracellular pathway may serve as an important therapeutic target for aberrant angiogenesis in exudative AMD.

Oral epithelial cells transplanted on to corneal surface tend to adapt to the ocular phenotype.

To understand the response of oral epithelial cells, transplanted on corneal surface to the ocular cues in vivo. The corneal button obtained after penetrating keratoplasty (PK) of an eye of a patient with total limbal stem cell deficiency (LSCD), previously treated with cultured oral mucosal epithelial transplantation (COMET) was examined by immunohistochemistry for the expression of keratins, p63, p75, PAX6, Ki-67, CD31, and CD34. COMET followed by optical-PK has improved visual acuity to 20/40 and rendered a stable ocular surface. The excised corneal tissue showed the presence of stratified epithelium with vasculatures. The epithelial cells of the corneal button expressed K3, K19, Ki-67, p63, p75 and the cornea-specific PAX6 and K12. This study confirms that the oral cells, transplanted to corneal surface, survive and stably reconstruct the ocular surface. They maintain their stemness at the ectopic site and acquire some of the corneal epithelial-like characters.

Coculture of autologous limbal and conjunctival epithelial cells to treat severe ocular surface disorders: long-term survival analysis.

BACKGROUND: Cultivated limbal epithelium for reconstruction of corneal surface is a well-established procedure; however, it is not adequate for damage which also extensively involves the conjunctiva. In severe cases of ocular surface damage that warrant additional conjunctival transplantation apart from cultivated limbal stem cell transplantation, we describe the long-term survival of a novel method of cocultivating autologous limbal and conjunctival epithelium on a single substrate. MATERIALS AND METHODS: Forty eyes of 39 patients with severe limbal stem cell deficiency and conjunctival scarring or symblepharon underwent transplantation of autologous cocultivated epithelium on human amniotic membrane. A ring barrier was used to segregate the central limbal and peripheral conjunctival epithelia in vitro. Patients were followed up at regular intervals to assess stability of the ocular surface, defined by absence of conjunctivalization into the central 4 mm of the cornea and absence of diffuse fluorescein staining. Penetrating keratoplasty (PKP) was subsequently performed, where indicated, in patients with surface stability. RESULTS: The cumulative survival probability was 60% at 1 year and 45% at 4 years by Kaplan-Meier analysis (mean follow-up duration: 33 ± 29 months, range: 1-87 months). Best-corrected visual acuity improved to greater than 20/200 in 38% eyes at the last follow-up, compared with 5% eyes before surgery. Immunohistochemistry in five of the corneal buttons excised for PKP showed an epithelial phenotype similar to cornea in all five. CONCLUSIONS: Synchronous use of cultured limbal and conjunctival epithelium offers a feasible alternative and a simpler one-step surgical approach to treat severe ocular surface disorders involving limbus and conjunctiva.

Derivation, characterization and retinal differentiation of induced pluripotent stem cells.

Millions of people world over suffer visual disability due to retinal dystrophies which can be age-related or a genetic disorder resulting in gradual degeneration of the retinal pigmented epithelial (RPE) cells and photoreceptors. Therefore, cell replacement therapy offers a great promise in treating such diseases. Since the adult retina does not harbour any stem cells, alternative stem cell sources like the embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer a great promise for generating different cell types of the retina. Here, we report the derivation of four iPSC lines from mouse embryonic fibroblasts (MEFs) using a cocktail of recombinant retroviruses carrying the genes for Oct4, Sox2, Klf4 and cMyc. The iPS clone MEF-4F3 was further characterized for stemness marker expression and stable reprogramming by immunocytochemistry, FACS and RT-PCR analysis. Methylation analysis of the nanog promoter confirmed the reprogrammed epigenetic state. Pluripotency was confirmed by embryoid body (EB) formation and lineage-specific marker expression. Also, upon retinal differentiation, patches of pigmented cells with typical cobble-stone phenotype similar to RPE cells are generated within 6 weeks and they expressed ZO-1 (tight junction protein), RPE65 and bestrophin (mature RPE markers) and showed phagocytic activity by the uptake of fluorescent latex beads.

Clinical outcomes of xeno-free allogeneic cultivated limbal epithelial transplantation for bilateral limbal stem cell deficiency.

PURPOSE: To report the clinical outcomes of allogeneic cell-based therapy for bilateral corneal blindness due to limbal stem cell deficiency (LSCD). METHODS: This retrospective study included 28 eyes of 21 patients, at least 8 years of age, with bilateral and total LSCD, treated between 2001 and 2010. A limbal biopsy was obtained from the eye of an adult living related donor. The limbal epithelial cells were cultivated in the laboratory using a xeno-free explant culture technique and transplanted onto the recipient eye after 10-14 days. All transplant recipients received topical and systemic immunosuppressants. RESULTS: At a mean follow-up of 4.8 ± 2.8 years, 20 (71.4%) eyes maintained a completely epithelised, avascular and stable corneal surface, and among them 13 (46.4%) eyes subsequently underwent a penetrating keratoplasty (PK). The Kaplan-Meier survival rate of the PK allograft was 76.9 ± 11.7% at 1 year with a median survival of 3.3 years. Visual acuity improved to 20/60 or better in 19 (67.8%) eyes. No donor or recipient eyes developed serious ocular complications. CONCLUSIONS: Allogeneic cultivated limbal epithelial transplantation, followed by PK when needed, can successfully restore the ocular surface and improve vision in patients with corneal blindness due to bilateral LSCD.

Clinical outcomes of xeno-free autologous cultivated limbal epithelial transplantation: a 10-year study.

PURPOSE: Ocular burns can damage the corneal epithelial stem cells located at the limbus. This study evaluated the efficacy of xeno-free autologous cell-based treatment of limbal stem cell deficiency. METHODS: This retrospective study included 200 patients, above 8 years of age, with clinically diagnosed unilateral total limbal stem cell deficiency due to ocular surface burns treated between 2001 and 2010. A small limbal biopsy was obtained from the unaffected eye. The limbal epithelial cells were expanded ex vivo on human amniotic membrane for 10-14 days using a xeno-free explant culture system. The resulting cultured epithelial monolayer and amniotic membrane substrate were transplanted on to the patient's affected eye. Postoperative corneal surface stability, visual improvement and complications were objectively analysed. RESULTS: A completely epithelised, avascular and clinically stable corneal surface was seen in 142 of 200 (71%) eyes at a mean follow-up of 3 ± 1.6 (range: 1-7.6) years. A two-line improvement in visual acuity, without further surgical intervention, was seen in 60.5% of eyes. All donor eyes remained healthy. CONCLUSIONS: Autologous cultivated limbal epithelial transplantation using a xeno-free explant culture technique was effective in long-term restoration of corneal epithelial stability and improvement of vision in eyes with ocular surface burns.

In vitro culture and expansion of human limbal epithelial cells.

Limbal stem cells (LSCs) have an important role in the maintenance of the corneal surface epithelium, and autologous cultured limbal epithelial cell transplantations have contributed substantially to the treatment of the visually disabling condition known as LSC deficiency. In this protocol, we describe a method of establishing human limbal epithelial cell cultures by a feeder-free explant culture technique using a small limbal biopsy specimen and human amniotic membrane (hAM) as the culture substrate. This protocol is free of animal-derived products and involves the use of human recombinant growth factors. In addition, the recombinant cell dissociation enzyme TrypLE is used to replace trypsin and autologous serum replaces FBS. It takes approximately 2 weeks to establish a confluent monolayer from which approximately 3 x 10(6) cells can be harvested. This procedure can be adopted for both basic research purposes and clinical applications.

A biomimetic scaffold for culturing limbal stem cells: a promising alternative for clinical transplantation.

Limbal tissues can be cultured on various types of scaffolds to create a sheet of limbal-corneal epithelium for research as well as clinical transplantation. An optically clear, biocompatible, biomimetic scaffold would be an ideal replacement graft for transplanting limbal stem cells. In this study, we evaluated the physical and culture characteristics of the recombinant human cross-linked collagen scaffold (RHC-III scaffold) and compared it with denuded human amniotic membrane (HAM). Optical/mechanical properties and microbial susceptibility were measured for the scaffolds. With the approval of the institutional review board, 2 mm fresh human limbal tissues were cultured on 2.5 x 2.5 cm(2) scaffolds in a medium containing autologous serum in a feeder cell-free submerged system. The cultured cell systems were characterized by morphology and immunohistochemistry for putative stem cells and differentiated cell markers. The refractive index (RI) and tensile strength of the RHC-III scaffold were comparable to human cornea, with delayed in vitro degradation compared to HAM. RHC-III scaffolds were 10-fold less susceptible to microbial growth. Cultures were initiated on day 1, expanded to form a monolayer by day 3 and covered the entire growth surface in 10 days. Stratified epithelium on the scaffolds was visualized by transmission electron microscopy. The cultured cells showed p63 and ABCG2 positivity in the basal layer and were immunoreactive for cytokeratin K3 and K12 in the suprabasal layers. RHC-III scaffold supports and retains the growth and stemness of limbal stem cells, in addition to resembling human cornea; thus, it could be a good replacement scaffold for growing cells for clinical transplantation.

Culture and characterization of oral mucosal epithelial cells on human amniotic membrane for ocular surface reconstruction.

PURPOSE: To culture oral mucosal epithelial cells on deepithelialized human amniotic membrane without the use of feeder cells and to compare the characteristics of cultured oral cells with cultured limbal and conjunctival epithelial cells for use in ocular surface reconstruction. METHODS: Oral biopsies were obtained from healthy volunteers after informed consent and were cultured on deepithelialized amniotic membrane for three to four weeks. Confluent cultures of limbal, oral, and conjunctival cells were subjected to characterization of markers of stem cells and of epithelial differentiation by reverse-transcription polymerase chain reaction (RT-PCR) and by immunohistochemistry. Ultrastructural studies were also performed using electron microscopy. RESULTS: A sheet of healthy, stratified oral epithelial cells was obtained within three to four weeks of culture. Electron microscopy demonstrated that the cells formed gap junctions and desmosomes. RT-PCR analysis showed that cultured oral epithelial cells expressed markers of epithelial differentiation such as cytokeratin K3, K4, K13, K15 and connexin 43. The cells also expressed stem cell markers of epithelial cells such as DeltaN isoforms of p63 as well as p75, a marker for stem cells of oral epithelium. The cells did not express cytokeratin K12 or Pax-6, an eye-specific transcription factor. CONCLUSIONS: Oral epithelial cells can be cultured as explants on deepithelialized amniotic membrane without using feeder cells. Characterization showed that these cells maintain the phenotypic characteristics of oral epithelial cells and that the culture is a heterogeneous population of differentiated cells and stem cells. We find the cultured oral epithelial cells usable for ocular surface reconstruction in patients having bilateral ocular surface diseases.