TGF-β 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-β signaling between EC and mural cells participates in vessel stabilization in vitro. We therefore investigated the role of TGF-β signaling in maintaining microvessel structure and function in the adult mouse retinal microvasculature. TGF-β 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-β 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-β 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-β signaling. These results implicate constitutive TGF-β signaling in maintaining the integrity and function of the adult microvasculature and shed light on the potential role of TGF-β 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

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.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.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. (C) 2008 Published by Elsevier Ltd

TGF-? 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-β signaling between EC and mural cells participates in vessel stabilization in vitro. We therefore investigated the role of TGF-β signaling in maintaining microvessel structure and function in the adult mouse retinal microvasculature. TGF-β 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-β 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-β 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-β signaling. These results implicate constitutive TGF-β signaling in maintaining the integrity and function of the adult microvasculature and shed light on the potential role of TGF-β signaling in vasoproliferative and vascular degenerative retinal diseases

Novel sutureless transplantation of bioadhesive-coated, freeze-dried amniotic membrane for ocular surface reconstruction. Invest Ophthalmol Vis Sci 48

PURPOSE. To evaluate the efficacy and safety of a novel sutureless transplantation of bioadhesive-coated, sterilized, freezedried 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 ␥-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. (Invest Ophthalmol Vis Sci

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

Novel sutureless transplantation freeze-dried amniotic membrane 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 FDAM. 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 FDAM. Consequently, this sutureless method of transplantation of FCFD-AM is safe, simple, and useful for ocular surface reconstruction

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

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

Inhibition of TGF-β decreases retinal perfusion and vascular autoregulation.

<p>After fourteen days of Ad-sEng and Ad-null expression, mice were injected with h.m.w. FITC-dextran through the left ventricle to localize perfused vessels. (A) Confocal analysis of retinal flat-mounts revealed reduced perfusion of the retina in the sEng-expressing mice compared to the control (representative photo of n = 12 mice). Scale bar = 200 µm. (B) The perfused vessels were visualized on retinal flat mounts by comparing the co-localization of type IV collagen (Cy3-red) and FITC and quantified on cryosections by comparing the number of vessels in the innermost vascular plexus (arrowheads) positive for both type IV collagen- and FITC to the number of vessels positive for type IV collagen but negative for FITC (C). (D) The retinas of sEng expressing mice show a marked reduction in the number of perfused vessels (n = 5, ** p<0.01). (E) After seven days of adenoviral expression, blood flow rates in the tail were measured non-invasively in response to intravascular injection of ACh. Measurements were made over 5 cycles pre-injection of ACh, normalized to 1 for each animal, and averaged at 5-cycle intervals post ACh injection. In Ad-null control mice, ACh increased tail vein blood flow rates 6–10 cycles post-injection, whereas blood flow rates were unchanged in Ad-sEng expressing mice (Ad-null: 1.619 µl/cycle, n = 5; Ad-sEng: 0.960 µl/cycle, n = 4, * p<0.01). Injection of 100 µl of saline in Ad-null or Ad-sEng mice elicited no response.</p