Heat treatment of retinal pigment epithelium induces production of elastic lamina components and antiangiogenic activity.

Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world. In advanced AMD, new vessels from choriocapillaris (CC) invade through the Bruch's membrane (BrM) into the retina, forming choroidal neovascularization (CNV). BrM, an elastic lamina that is located between the retinal pigment epithelium (RPE) and CC, is thought to act as a physical and functional barrier against CNV. The BrM of patients with early AMD are characterized by decreased levels of antiangiogenic factors, including endostatin, thrombospondin-1 (TSP-1), and pigment epithelium-derived factor (PEDF), as well as by degeneration of the elastic layer. Motivated by a previous report that heat increases elastin expression in human skin, we examined the effect of heat on human ARPE-19 cell production of BrM components. Heat treatment stimulated the production of BrM components, including TSP-1, PEDF, and tropoelastin in vitro and increased the antiangiogenic activity of RPE measured in a mouse corneal pocket assay. The effect of heat on experimental CNV was investigated by pretreating the retina with heat via infrared diode laser prior to the induction of CNV. Heat treatment blocked the development of experimental CNV in vivo. These findings suggest that heat treatment may restore BrM integrity and barrier function against new vessel growth.

An essential role for RPE-derived soluble VEGF in the maintenance of the choriocapillaris.

Clinical and experimental observations indicate a role for VEGF secreted by the retinal pigment epithelium (RPE) in the maintenance of the choriocapillaris (CC). VEGF in mice is produced as three isoforms, VEGF120, VEGF164, and VEGF188, that differ in their ability to bind heparan sulfate proteoglycan. RPE normally produces the more soluble isoforms, VEGF120 and VEGF164, but virtually no VEGF188, reflecting the fact that molecules secreted by the RPE must diffuse across Bruch's membrane (BrM) to reach the choriocapillaris. To determine the role of RPE-derived soluble VEGF on the choriocapillaris survival, we used mice that produce only VEGF188. VEGF188/188 mice exhibited normal choriocapillaris development. However, beginning at 7 months of age, we observed a progressive degeneration characterized by choriocapillaris atrophy, RPE and BrM abnormalities, culminating in areas of RPE loss and dramatic choroidal remodeling. Increased photoreceptor apoptosis in aged VEGF188/188 mice led to a decline in visual acuity as detected by electroretinogram (ERG). These changes are reminiscent of geographic atrophy (GA) and point to a role for RPE-derived VEGF in the maintenance of the choriocapillaris.

TGF-beta is required for vascular barrier function, endothelial survival and homeostasis of the adult microvasculature.

Pericyte-endothelial cell (EC) interactions are critical to both vascular development and vessel stability. We have previously shown that TGF-beta signaling between EC and mural cells participates in vessel stabilization in vitro. We therefore investigated the role of TGF-beta signaling in maintaining microvessel structure and function in the adult mouse retinal microvasculature. TGF-beta signaling was inhibited by systemic expression of soluble endoglin (sEng) and inhibition was demonstrated by reduced phospho-smad2 in the adult retina. Blockade of TGF-beta signaling led to increased vascular and neural cell apoptosis in the retina, which was associated with decreased retinal function, as measured by electroretinogram (ERG). Perfusion of the inner retinal vasculature was impaired and was accompanied by defective autoregulation and loss of capillary integrity. Fundus angiography and Evans blue permeability assay revealed a breakdown of the blood-retinal-barrier that was characterized by decreased association between the tight junction proteins zo-1 and occludin. Inhibition of TGF-beta signaling in cocultures of EC and 10T1/2 cells corroborated the in vivo findings, with impaired EC barrier function, dissociation of EC from 10T1/2 cells, and endothelial cell death, supporting the role of EC-mesenchymal interactions in TGF-beta signaling. These results implicate constitutive TGF-beta signaling in maintaining the integrity and function of the adult microvasculature and shed light on the potential role of TGF-beta signaling in vasoproliferative and vascular degenerative retinal diseases.

Endogenous VEGF is required for visual function: evidence for a survival role on müller cells and photoreceptors.

BACKGROUND: Vascular endothelial growth factor (VEGF) is well known for its role in normal and pathologic neovascularization. However, a growing body of evidence indicates that VEGF also acts on non-vascular cells, both developmentally as well as in the adult. In light of the widespread use of systemic and intraocular anti-VEGF therapies for the treatment of angiogenesis associated with tumor growth and wet macular degeneration, systematic investigation of the role of VEGF in the adult retina is critical. METHODS AND FINDINGS: Using immunohistochemistry and Lac-Z reporter mouse lines, we report that VEGF is produced by various cells in the adult mouse retina and that VEGFR2, the primary signaling receptor, is also widely expressed, with strong expression by Müller cells and photoreceptors. Systemic neutralization of VEGF was accomplished in mice by adenoviral expression of sFlt1. After 14 days of VEGF neutralization, there was no effect on the inner and outer retina vasculature, but a significant increase in apoptosis of cells in the inner and outer nuclear layers. By four weeks, the increase in neural cell death was associated with reduced thickness of the inner and outer nuclear layers and a decline in retinal function as measured by electroretinograms. siRNA-based suppression of VEGF expression in a Müller cell line in vitro supports the existence of an autocrine role for VEGF in Müller cell survival. Similarly, the addition of exogenous VEGF to freshly isolated photoreceptor cells and outer-nuclear-layer explants demonstrated VEGF to be highly neuroprotective. CONCLUSIONS: These results indicate an important role for endogenous VEGF in the maintenance and function of adult retina neuronal cells and indicate that anti-VEGF therapies should be administered with caution.

The use of trehalose-treated freeze-dried amniotic membrane for ocular surface reconstruction.

The aim of this study was to evaluate the efficacy and safety of trehalose-treated freeze-dried amniotic membrane (TT-FDAM) for ocular surface reconstruction. Human AM deprived of amniotic epithelial cells was first incubated with 10% trehalose solution, and then freeze-dried, vacuum-packed, and sterilized with gamma-irradiation. The resultant newly developed TT-FDAM was characterized for its physical, biological, and morphological properties by comprehensive physical assays, immunohistochemistry, electron microscopy, cell adhesion assay, 3D cell culture, and an in vivo biocompatibility test. The adaptability of TT-FDAM was markedly improved as compared to FDAM. Immunohistochemistry for several extracellular matrix molecules revealed that the process of freeze-drying and irradiation apparently did not affect its biological properties, however, electron microscopy revealed that the detailed morphological appearance of TT-FDAM is more similar to that of native AM than to FDAM. Intracorneal and scleral-surface transplantation of TT-FDAM showed excellent biocompatibility with ocular surface tissues. Thus, TT-FDAM retained most of the physical, biological, and morphological characteristics of native AM, consequently it is a useful biomaterial for ocular surface reconstruction.

Hes1 regulates corneal development and the function of corneal epithelial stem/progenitor cells.

Hes1, a major target gene in Notch signaling, regulates the fate and differentiation of various cell types in many developmental systems. To gain a novel insight into the role of Hes1 in corneal tissue, we performed gain-of-function and loss-of-function studies. We show that corneal development was severely disturbed in Hes1-null mice. Hes1-null corneas manifested abnormal junctional specialization, cell differentiation, and less cell proliferation ability. Worthy of note, Hes1 is expressed mainly in the corneal epithelial stem/progenitor cells and is not detected in the differentiated corneal epithelial cells. Expression of Hes1 is closely linked with corneal epithelial stem/progenitor cell proliferation activity in vivo. Moreover, forced Hes1 expression inhibits the differentiation of corneal epithelial stem/progenitor cells and maintains these cells' undifferentiated state. Our data provide the first evidence that Hes1 regulates corneal development and the homeostatic function of corneal epithelial stem/progenitor cells.

Novel sutureless transplantation of bioadhesive-coated, freeze-dried amniotic membrane for ocular surface reconstruction.

PURPOSE: To evaluate the efficacy and safety of a novel sutureless transplantation of bioadhesive-coated, sterilized, freeze-dried amniotic membrane (FD-AM) for ocular surface reconstruction. METHODS: A bioadhesive-coated, freeze-dried amniotic membrane was made by freeze drying the denuded AM in a vacuum, applying the minimum amount of fibrin glue (mixture of fibrinogen and thrombin) necessary to retain adhesion on the chorionic side, and sterilizing it by gamma-radiation. The resultant AM was characterized for its biological and morphologic properties by immunohistochemical and electron microscopic examination. In addition, fibrin glue-coated, freeze-dried (FCFD) AM was transplanted onto a rabbit scleral surface without sutures, to examine its biocompatibility. RESULTS: Immunohistochemistry of the FCFD-AM revealed that fibrinogen existed on its chorionic side, and the process of applying fibrin glue did not affect its biological and morphologic properties. Moreover, electron microscopic examination of the chorionic side of the FCFD-AM revealed tiny microfibrils (which are probably fibrinogen protofibrils), and showed that the epithelial surface of FCFD-AM consisted of intact basal lamina similar to that of FD-AM. FCFD-AM transplantation was very easily performed, and the graft adhered to the bare sclera immediately. Though the fibrinogen naturally biodegraded within 2 weeks, the FCFD-AM remained for at least 12 weeks after transplantation. Epithelialization on the FCFD-AM was achieved within 2 weeks, as was the case with FD-AM transplantation. The conjunctival epithelium on the FCFD-AM was well stratified and not keratinized, suggesting that FCFD-AM supports normal cell differentiation. CONCLUSIONS: The FCFD-AM retained most of the biological characteristics of FD-AM. Consequently, this sutureless method of transplantation of FCFD-AM is safe, simple, and useful for ocular surface reconstruction.

Novel clinical application of sterilized, freeze-dried amniotic membrane to treat patients with pterygium.

PURPOSE: To evaluate the use of sterilized, freeze-dried amniotic membrane (FD-AM) transplantation for pterygium surgery. METHODS: This study involved a prospective, non-comparative, interventional case series. Thirteen eyes of 13 patients with primary (eight eyes) or recurrent (five eyes) pterygium were studied. After excision of the pterygium fibrous tissues and application of intraoperative use of mitomycin-C, sterilized FD-AM was sutured over the bare scleral defect. The integrity of the FD-AM graft, epithelialization over the FD-AM, pterygium recurrence and postoperative complications were evaluated. RESULTS: Postoperatively, the FD-AM was well retained in all patients, and complete epithelialization over the transplanted membrane was achieved within 1-2 weeks. All patients demonstrated early resolution of ocular inflammation and there was no recurrence of pterygium in any of the treated patients during the mean follow-up of 13.9 +/- 6.0 months. No ocular complications were noted following transplantation. CONCLUSION: Sterilized FD-AM showed excellent biocompatibility on the human ocular surface. This novel and promising biomaterial may be a useful alternative to conjunctival grafting in the treatment of pterygium.

Different expression of angiogenesis-related factors between human cultivated corneal and oral epithelial sheets.

We developed a cultivated oral mucosal epithelial sheet (COE) transplantation system to address severe human ocular surface disorders. Unlike the cultivated corneal epithelial sheet (CCE), the COE induces mild superficial peripheral neovascularization although central clarity is maintained. To evaluate the characteristic differences between CCE and COE regarding to angiogenesis, we examined the expression of angiogenesis-related factors in CCE and COE. Using samples of CCE and COE, we immunohistochemically determined protein expression of the angiogenesis related factors: Thrombospondin-1 (TSP-1), pigment epithelium derived factor (PEDF), endostatin, angiostatin, vascular endothelial growth factor (VEGF), Fms-like tyrosine kinase 1 (Flt-1), kinase insert domain receptor (KDR), and basic fibroblast growth factor (bFGF). We used Western blot analysis to confirm the factors that were immunohistochemically different in CCE and COE. The immunohistochemical staining intensity of TSP-1 was higher in CCE than COE and by Western blot analysis the expression of TSP-1 was significantly higher in CCE than COE (P<0.05). PEDF and endostatin stained moderately stronger in CCE than COE. Immunohistochemically there was no obvious difference between CCE and COE with respect to angiostatin, VEGF, Flt-1, KDR, and bFGF. In comparison with CCE, COE showed decreased expression of anti-angiogenic factors particularly TSP-1. This different expression may relate to the superficial peripheral neovascularization encountered after COE transplantation.

Unique distribution of thrombospondin-1 in human ocular surface epithelium.

PURPOSE: The study was conducted to elucidate the detailed expression pattern of angiogenesis-related factors in human ocular surface epithelium. The focus was factors with significantly higher gene expression in corneal epithelium (CE) than in conjunctival epithelium (CJE). METHODS: The relative gene expression of 36 angiogenesis-related factors was compared in human CE and CJE, by using the introduced amplified fragment-length polymorphism (iAFLP) METHOD: Also examined were the expression patterns in the CE, limbal epithelium (LE), and CJE of factors with significantly higher expression in the CE, by using real-time PCR, in situ hybridization, immunohistochemistry, and immunoelectron microscopy. RESULTS: Only thrombospondin (TSP)-1 exhibited significantly higher expression in the CE. In situ hybridization and real-time PCR showed TSP-1 transcripts in the basal cells of the CE and LE. Compared with the CJE, they were significantly upregulated at those sites. Immunohistochemistry revealed that TSP-1 was strongly expressed in the basal region of the CE. Its expression was faint in LE and absent in CJE. Immunoelectron microscopy revealed that the CE and LE demonstrated TSP-1 labeling just below the epithelium, in the basal region of basal cells, and occasionally in the basal cell membrane. There was little or no labeling in the CJE. CONCLUSIONS: In the human ocular surface epithelium, basal cells of the CE and LE, but not of the CJE, synthesize TSP-1. High levels of TSP-1 are present only just below the CE. Its unique distribution may be related to corneal avascularity and integrity.

The use of autologous serum in the development of corneal and oral epithelial equivalents in patients with Stevens-Johnson syndrome.

PURPOSE: To evaluate the use of autologous serum (AS) from patients with severe ocular surface disease (OSD) in the development of transplantable corneal and oral epithelial tissue equivalents and to compare it with the use of conventional culture methods by using fetal bovine serum (FBS). METHODS: AS was obtained from patients with severe OSD secondary to Stevens-Johnson syndrome. Corneal and oral epithelial cells were cultivated in medium supplemented with either AS or FBS. Corneal and oral epithelial equivalents were constructed on denuded amniotic membranes. The bromodeoxyuridine (BrdU) ELISA cell proliferation assay and colony-forming efficiency (CFE) of cells cultivated in AS- or FBS-supplemented media were compared. The morphologic characteristics and the basement membrane assembly of cultivated epithelial equivalents were analyzed by light and electron microscopy, as well as by immunohistochemistry. RESULTS: BrdU proliferation assay and CFE analysis showed that human corneal and oral epithelial cells cultivated in AS-supplemented media had comparable proliferative capacities compared with FBS-supplemented media. The corneal and oral epithelial equivalents cultivated in AS- and FBS-supplemented media were morphologically similar and demonstrated the normal expression of tissue-specific keratins and basement membrane assembly. The presence of a well-formed stratified epithelium, a basement membrane, and hemidesmosomal attachments was confirmed by electron microscopy. CONCLUSIONS: AS-supplemented cultures were effective in supporting the proliferation of human corneal and oral epithelial cells, as well as the development of transplantable epithelial equivalents. The use of AS is of clinical importance in the development of autologous xenobiotic-free bioengineered ocular surface equivalents for clinical transplantation.