Retrocorneal Membrane Formation Research Articles

ZEB1 Mediates Fibrosis in Corneal Endothelial Mesenchymal Transition Through SP1 and SP3.

PurposeZEB1 is induced during endothelial-mesenchymal transition (EnMT) in the cornea. Induction of SP1 and SP3 by ZEB1 along with identification of putative SP1 and SP3 binding sites in promoters of EnMT-associated gene lead us to investigate their roles in retrocorneal membrane formation in the corneal endothelium.MethodsExpressions of SP1, SP3, and EnMT associated genes were analyzed by immunoblotting and semiquantitative reverse transcription polymerase chain reaction. Accell SMARTpool siRNAs targeting ZEB1, SP1, and SP3 were used for gene knockdown. SP1 and SP3 binding to promoters of EnMT associated genes was investigated by chromatin immunoprecipitation assay. Corneal endothelium in mice was surgically injured in vivo under direct visualization.ResultsTransient Fibroblast Growth Factor 2 stimulation increased the expression of both SP1 and SP3 in the human corneal endothelium ex vivo. ZEB1 siRNA knockdown inhibited FGF2-induced SP1 mRNA and protein but not the expression of SP3. FGF2-induced expression of EnMT-related genes, such as fibronectin, vimentin, and type I collagen, was reduced by both SP1 and SP3 siRNA knockdown, with inhibition of SP1 having a greater inhibitory effect than SP3. Additionally, although SP1 and SP3 proteins were found to bind together, SP1 and SP3 could bind to the same promoter binding sites of EnMT-related genes in the absence of the other. Moreover, siRNA knockdown of Zeb1 inhibited injury-dependent RCM formation in mouse corneal endothelium in vivo.ConclusionsZeb1, through SP1 and SP3, plays a central role in mesenchymal transition induced fibrosis in the corneal endothelium and suggests that Zeb1 could be targeted to inhibit anterior segment fibrosis.

Fibroblast growth factor 2 induces proliferation and fibrosis via SNAI1-mediated activation of CDK2 and ZEB1 in corneal endothelium

Investigating stimulation of endogenous wound healing in corneal endothelial cells (CECs) may help address the global shortage of donor corneas by decreasing the number of transplants performed for blindness because of endothelial dysfunction. We previously reported that IL-1β stimulation leads to fibroblast growth factor (FGF2) expression, enhancing migration and proliferation of mammalian CECs. However, FGF2 also promotes the endothelial-mesenchymal transition, which can lead to retrocorneal membrane formation and blindness. This prompted us to investigate downstream FGF2 signaling targets that could be manipulated to prevent retrocorneal membrane formation. FGF2 stimulation altered cell morphology and induced expression of mesenchymal transition marker genes such as snail family transcriptional repressor 1 (SNAI1), SNAI2, zinc finger E-box-binding homeobox 1 (ZEB1), and ZEB2 This, in turn, induced expression of fibronectin, vimentin, and type I collagen, and suppressed E-cadherin in CECs in vitro and ex vivo siRNA-mediated SNAI1 knockdown revealed that SNAI1 induces ZEB1 expression, in turn inducing expression of type I collagen, the major component of retrocorneal membranes, and of cyclin-dependent kinase 2 (CDK2) and cyclin E1, promoting cell proliferation. siRNA-mediated knockdown of SNAI1 or ZEB1, but not of CDK2, inhibited FGF2-dependent expression of fibronectin, vimentin, and type I collagen and of suppression of E-cadherin expression. We conclude that SNAI1 is a key regulator of FGF2-dependent mesenchymal transition in human ex vivo corneal endothelium, with ZEB1 regulating type I collagen expression and CDK2 regulating cell proliferation. These results suggest that SNAI1 promotes fibrosis and cell proliferation in human corneal endothelium through ZEB1 and CDK2.

Mesenchymal stem cell therapy for retro-corneal membrane - A clinical challenge in full-thickness transplantation of biosynthetic corneal equivalents.

The manuscript addresses the potential value of bone marrow-derived mesenchymal stem cell therapy for retro-corneal membrane (RCM) formation in full-thickness transplantation of biosynthetic corneal equivalents. This work reports the pathophysiological changes in the anterior chamber of the mouse eye following full-thickness recombinant human cross-linked collagen-based hydrogel implants in which persistent fibrin promotes the development of dense RCM. Furthermore, pre-treatment with mesenchymal stem cells reduces RCM formation and enhances corneal transparency.

Retrocorneal Membrane After Descemet Membrane Endothelial Keratoplasty

To describe a retrocorneal membrane after Descemet membrane endothelial keratoplasty (DMEK). Case report and review of the medical literature. A 73-year-old woman diagnosed with bilateral pseudophakic bullous keratopathy underwent DMEK in her left eye. Her preoperative best-spectacle-corrected visual acuity OD was 20/500 and OS was 20/500. After DMEK, graft detachment was found on the first and second postoperative days. Graft repositioning with air was performed 2 times. One month after DMEK, a membranous structure was identified behind the cornea by slit-lamp examination. The patient was applying topical levofloxacin 0.5% (Cravit; Santen, Japan) and prednisolone acetate 1% (Pred Forte; Allergan, Irvine, CA) 4 times per day. Two months after DMEK, the retrocorneal membrane disappeared, and the patient's best-corrected visual acuity OS improved to 20/40. We report the first case of retrocorneal membrane formation after DMEK surgery.

Notch Signal Regulates Corneal Endothelial-to-Mesenchymal Transition

Endothelial-to-mesenchymal transition (EnMT) is a cell transformation process involved in both morphogenesis and pathogenesis. EnMT of corneal endothelial cells happens after endothelial injury and during ex vivo culture. Previous studies have shown that the transforming growth factor-β signaling pathway is involved in this transition. In this study, we found that rat corneal endothelial cells could spontaneously undergo EnMT during ex vivo culture. This change in rat corneal endothelial cells was associated with Notch signaling pathway activation after the first passage, which was blocked by the Notch inhibitor N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). This inhibitor also prevented transforming growth factor β1-, β2-, and β3-induced EnMT and reversed transformed rat corneal endothelial cells to a normal phenotype. Furthermore, DAPT treatment blocked retrocorneal membrane formation in a rat corneal endothelium damage model. Our study indicates that the Notch signaling pathway is involved in the corneal EnMT process, which may be a novel therapeutic target for treating corneal endothelial fibrogenic disorders.

Failed Descemet-Stripping Automated Endothelial Keratoplasty Grafts: A Clinicopathologic Analysis

To describe the clinicopathologic findings in failed Descemet-stripping automated endothelial keratoplasty (DSAEK) grafts. Retrospective, interventional case series. New York Eye and Ear Infirmary. Twenty-one patients with 22 failed DSAEK grafts treated between March 1, 2006 and February 1, 2008. Repeat DSAEK or penetrating keratoplasty were performed in the eyes with failed grafts. All failed grafts were examined histopathologically. Histopathologic parameters studied in failed DSAEK grafts included endothelial cell count, interface characteristics, retrocorneal membrane formation, inflammation, and immunoreactivity for herpes simplex virus type 1 (HSV-1) antigen. DSAEK failure was strongly associated with postoperative lenticle dislocation. Graft failure was primary in 19 DSAEKs and secondary to rejection, eccentric trephination with epithelial ingrowth, or bacterial infection in the remaining 3. All failed grafts demonstrated endothelial hypocellularity and stromal edema. Additional findings included stromal inflammation (68%), interface fibrosis (50%), retrocorneal membrane (36%), unplanned retention of Descemet membrane (14%), immunoreactivity for HSV-1 (14%), paucicellular stroma (14%), and uneven trephination with epithelial ingrowth (5%). Most DSAEK failures are secondary to endothelial cell loss. Other contributing factors include interface fibrosis, retrocorneal membrane formation, retained host Descemet membrane, uneven trephination, epithelial ingrowth, graft rejection, and infection.

Histological changes and wound healing response following noncontact holmium: YAG laser thermal keratoplasty

To evaluate acute histological changes and the induced wound healing response in corneal tissue following noncontact holmium:YAG laser thermal keratoplasty (LTK).LTK using 10 pulses and a range of radiant energies was performed on 3 human corneas one day prior ro their removal at penetrating keratoplasty. Rabbit corneas were treated with 10-pulse and 5-pulse LTK and followed for up to 3 months. Tissues were studies with light and transmission electron microscopy and immunohistochemistry.The amount of acute tissue injury increased with increasing pulse radiant energy. In human corneas, changes in the irradiated zones included epithelial cell injury and death loss of fine filamentous structure in Bowman's layer, disruption of stromal lamallae, and keratocyte injury and death. In the rabbit corneas, similar acute changes were noted. By 3 weeks, epithelial hyperplasia and stromal contraction were present. Wound healing in the rabbit corneas included repair of the epithelial attachment complex, keratocyte activation, synthesis of type I collagen, partial restoration of stromal keratan sulfate and type VI collagen, and retrocorneal membrane formation. Compared to 10-pulse treatments, 5-pulse treatments produced less acute tissue injury and had more rapid restoration of normal stromal architecture.Noncontact LTK produces acute epithelial and stromal tissue changes and in rabbit corneas stimulates a brisk wound healing response. These changes could contribute to postoperative regression of induced refractive correction. Further work is required to determine if reductions in the magnitude of acute tissue injury and induced wound healing response will enhance the efficacy and stability of LTK.

Histologic changes and wound healing response following 10-pulse noncontact holmium:YAG laser thermal keratoplasty.

Noncontact holmium:YAG laser thermal keratoplasty (Ho:YAG LTK) is a promising new technology for correction of hyperopia and astigmatism. We studied the acute histologic changes and wound healing response following Ho:YAG LTK performed with treatment parameters encompassing those used in clinical studies. We performed 10-pulse noncontact Ho:YAG LTK on three human corneas 1 day before their removal at penetrating keratoplasty and on six New Zealand white rabbit corneas followed for up to 3 months. Tissues were studied with light and transmission electron microscopy and immunohistochemistry. The amount of acute tissue injury increased according to the pulse radiant energy. In human corneas, changes in the irradiated zones included epithelial cell injury and death, loss of fine filamentous structure in Bowman's layer, disruption of stromal lamellae, and keratocyte injury and death. In the rabbit corneas, similar acute changes were noted. By 3 weeks, epithelial hyperplasia and stromal contraction were present. Wound healing in the rabbits included repair of the epithelial attachment complex, keratocyte activation, synthesis of type I collagen, partial restoration of stromal keratan sulfate and type VI collagen, and retrocorneal membrane formation. Noncontact Ho:YAG LTK produces acute epithelial and stromal tissue changes and in rabbit corneas stimulates a brisk wound healing response.

Transplantation of corneal endothelial cells using a cell carrier device.

Penetrating keratoplasty is currently the only treatment for corneal endothelial dysfunction. Although corneal transplantation has a high success rate, a few problems still remain, such as the limited availability of donor grafts, the change in refraction after penetrating keratoplasty, and the higher chance of immune rejection. In this study, a coated hydrogel lens (Chiron Ophthalmics Inc., Irvine CA, U.S.A.) has been used as a carrier to transplant cultured homologous kitten and rabbit corneal endothelial cells into adult cats and rabbits. The transplantation procedure was the same in both species. Corneal endothelial cells from homologous rabbits or cats were seeded on coated hydrogel lenses and cultured until they reached a complete monolayer with an average cell density of 2,500 cells/mm2. Five weeks before transplantation surgery, corneal endothelial cells were scraped to induce corneal edema. The cell carrier device was then transplanted as follows: a trephine cut (7.7 mm) was made into the stroma, producing an outer corneal plug. The inner cornea was then cut by using a 5.5-mm trephine, and this inner plug was discarded. The implant was inserted and the outer corneal plug was sutured back into place. Corneas cleared completely within 3 days in both rabbits and cats, and stayed clear for an average of 40 days in rabbits and 50 days in cats. The histopathological evaluation of the rejected grafts showed vascularized retrocorneal membrane formation in cats, whereas in rabbits severe cellular infiltration of the stroma with neovascularization occurred without retrocorneal membrane formation.

Histopathology of Silicone Oil Keratopathy in Humans

Corneal endothelial decompensation is a frequent complication when silicone oil is used as a tamponade following vitrectomy for treatment of proliferative vitreoretinopathy in an aphakic eye. We evaluated the clinical, histopathological, and ultrastructural features of silicone oil-induced keratopathy in 10 patients who developed corneal complications requiring penetrating keratoplasty. Clinically, some cases showed corneal edema, corneal hypesthesia, endothelial opacification, band keratopathy, and peripheral corneal vascularization. Histopathologically, retrocorneal membranes were present, and different degrees of stromal hypercellularity, superficial stromal calcification, and vascularization were noted. The ultrastructural features of endothelial cell loss and retrocorneal membrane formation are consistent with changes previously reported in rabbits and cats receiving intracameral silicone oil injections.

Histopathology of Silicone Oil Keratopathy in Humans

Corneal endothelial decompensation is a frequent complication when silicone oil is used as a tamponade following vitrectomy for treatment of proliferative vitreoretinopathy in an aphakic eye. We evaluated the clinical, histopathological, and ultrastructural features of silicone oil-induced keratopathy in 10 patients who developed corneal complications requiring penetrating keratoplasty. Clinically, some cases showed corneal edema, corneal hypesthesia, endothelial opacification, band keratopathy, and peripheral corneal vascularization. Histopathologically, retrocorneal membranes were present, and different degrees of stromal hypercellularity, superficial stromal calcification, and vascularization were noted. The ultrastructural features of endothelial cell loss and retrocorneal membrane formation are consistent with changes previously reported in rabbits and cats receiving intracameral silicone oil injections.

Penetrating Keratoplasty-Reply

<h3>In Reply.</h3> —Dr Morrison and I were aware of the article cited by Dr Waring, and, in addition, we reviewed the excellent and comprehensive report on normal and pathologic structure and function of the corneal endothelium that he presented to the American Academy of Ophthalmology in 1981. Accordingly, we did consider that the splitting of the posterior wound edges observed in three (not 15 as stated by Dr Waring) of our 23 postkeratoplasty specimens actually might have occurred between Descemet's membrane and an acellular posterior collagenous layer; however, judging by thickness, staining characteristics and tracing of Descemet's membrane through multiple sections of the same specimen, we were confident, even with light microscopy, that in these three specimens the splitting occurred between the thinner anterior and the thicker posterior layers of Descemet's membrane. We have identified various types, stages, and degrees of retrocorneal membrane formation in other specimens and are familiar

Evaluation of Through-and-Through Corneal Sutures

Sutures placed at the level of Descemet's membrane were compared with sutures placed through and through the full corneal thickness in 42 rabbit corneas using cataract and corneal wounds. The rabbits were killed, and sutures were removed at various postoperative periods up to four weeks. Evaluation of the wounds demonstrated that although the through-and-through technique provided good wound closure in the immediate postoperative period, the technique produced more damage to Descemet's membrane and to the endothelial cells, both at the time of suturing and at the time of suture removal, and tended to produce greater retrocorneal membrane formation. In addition, the through-and-through suturing technique was technically more difficult. In this study performed in rabbits, the one advantage of the through-and-through suture, ie, immediate posterior wound closure, was outweighed by its many disadvantages.

Use of platelet-fibrinogen-thrombin mixture to seal experimental penetrating corneal wounds

A platelet-fibrinogen-thrombin mixture utilizing autologous platelets was studied for its potential to seal perforating corneal wounds. In rabbits, the mixture demonstrated sufficient adhesive properties to permit 75% of penetrating keratoplasties to remain in place without the use of sutures. All 12 grafts held initially; after 48 h three grafts extruded. In control eyes only two out of 12 (16%) of the corneal grafts remained in place. The material is simple to prepare and apply and is not toxic to the cornea. It does not cause inflammation or lid irritation. Intraocular complications observed with its use were transient anterior synechiae and retrocorneal membrane formation.

Endothelial rejection in human transplants.

The clinical appearance of endothelial rejection following penetrating keratoplasty was studied in three human cases. On the posterior surface of each graft was an endothelial rejection line formed by keratitic precipitates. In two cases, the rejection process was reversed with corticosteroids, while in one case, the endothelial rejection line progressed across the back of the graft, destroying the transplanted endothelium. This latter graft became available for study five weeks after the onset of rejection. Routine histologic and scanning electron microscopic examination of this specimen showed nearly complete destruction of endothelial cells and early retrocorneal membrane formation. The presence of an endothelial rejection line proved a common helpful sign in the diagnosis of early graft rejection.