Effect of N-oleoyl dopamine on myofibroblast trans-differentiation of retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells contribute to several clinical conditions resulting in retinal fibrotic scars. Myofibroblast trans-differentiation of RPE cells is a critical step in the process of retinal fibrosis. In this study, we investigated the effects of N-oleoyl dopamine (OLDA), a newer endocannabinoid with a structure distinct from classic endocannabinoids, on TGF-ß2-induced myofibroblast trans-differentiation of porcine RPE cells. Using an in vitro collagen matrix contraction assay, OLDA was found to inhibit TGF-ß2 induced contraction of collagen matrices by porcine RPE cells. This effect was concentration-dependent, with significant inhibition of contraction observed at 3 µM and 10 µM. OLDA did not affect the proliferation of porcine RPE cells. Immunocytochemistry showed that at 3 µM, OLDA decreased incorporation of α-SMA in the stress fibers of TGF-ß2-treated RPE cells. In addition, western blot analysis showed that 3 µM OLDA significantly downregulated TGF-ß2-induced α-SMA protein expression. Taken together these results demonstrate that OLDA inhibits TGF-ß induced myofibroblast trans-differentiation of RPE cells. It has been established that classic endocannabinoid such as anandamide, by activating the CB1 cannabinoid receptor, promote fibrosis in multiple organ systems. In contrast, this study demonstrates that OLDA, an endocannabinoid with a chemical structure distinct from classic endocannabinoids, inhibits myofibroblast trans-differentiation, an important step in fibrosis. Unlike classic endocannabinoids, OLDA has weak affinity for the CB1 receptor. Instead, OLDA acts on non-classic cannabinoid receptors such as GPR119, GPR6, and TRPV1. Therefore, our study indicates that the newer endocannabinoid OLDA and its non-classic cannabinoid receptors could potentially be novel therapeutic targets for treating ocular diseases involving retinal fibrosis and fibrotic pathologies in other organ systems.

Sustained dasatinib treatment prevents early fibrotic changes following ocular trauma.

PURPOSE: Posterior ocular trauma and the subsequent fibrotic retinal complication termed proliferative vitreoretinopathy (PVR) are leading causes of blindness in children and young adults. A previous study suggested that changes occurring within the first month post-trauma can lead to development of PVR later. The aim of this study was to examine the effect of dasatinib, a tyrosine kinase inhibitor clinically used to treat chronic myeloid leukemia, on fibrotic changes occurring within the first month following ocular trauma. METHODS: A previously established swine ocular trauma model that mimics both contusion and penetrating injuries was used. Dasatinib was administered on days 4 and 18 post-trauma via intravitreal injection of either bolus solution or suspension of a sustained release system incorporated in biodegradable poly (lactic-co-glycolic acid) (PLGA) nanoparticles. Animals were followed up to day 32, and the development of traction full-thickness fold in the posterior retina was assessed. RESULTS: A full-thickness retinal fold extending from the wound site developed in 3 out of 4 control eyes injected with PLGA nanoparticles alone at 1 month. Administration of dasatinib solution had little preventative effect with 6 out of 7 eyes developing a fold. In contrast, dasatinib-incorporated PLGA nanoparticle injection significantly reduced the incidence of fold to 1 out of 10 eyes. CONCLUSIONS: Injection of dasatinib-incorporated PLGA significantly reduced early fibrotic retinal changes which eventually lead to PVR following posterior ocular trauma. Thus, our sustained dasatinib release system can potentially be used to both prevent and/or broaden the surgical treatment window for PVR.

Focal adhesion kinase family is involved in matrix contraction by transdifferentiated Müller cells.

Transdifferentiated Müller cells that adopt a fibroblastic/myofibroblastic phenotype have been identified in epiretinal membranes (ERMs) in several ocular disorders, and have been implicated to play a role in the formation and/or the contraction of ERMs. We have previously demonstrated that dasatinib, a dual inhibitor of Src-family kinases and Abl kinase, can prevent matrix contraction by transdifferentiated Müller cells. In this study, we examined molecules involved in matrix contraction downstream of primary dasatinib targets. Tyrosine phosphorylation of focal adhesion kinase (FAK) family members FAK and PYK2 was significantly reduced by dasatinib, and select inhibitors for these kinases PF431396, which inhibits both FAK and PYK2, and PF573228, which only inhibits FAK and not PYK2, significantly reduced matrix contraction by transdifferentiated Müller cells. Dasatinib and PF431396 significantly reduced phosphorylation of Hic-5, a protein implicated to play a role in focal adhesions and cell signaling. Our data shows that FAK family members are involved in matrix contraction by transdifferentiated Müller cells, and also implicates that Hic-5 is situated downstream of the FAK family within the signaling pathway.

Role of epithelial-mesenchymal transition in proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is a potentially blinding fibrotic complication. It is caused by the formation and contraction of epiretinal membranes (ERMs) that ultimately lead to retinal folds and traction retinal detachments. While multiple cell types have been identified in ERMs, retinal pigment epithelial (RPE) cells have long been implicated as a key player in the pathophysiology of PVR. Clinical and experimental evidence has shown that RPE cells undergo epithelial-mesenchymal transition (EMT) to adopt a fibroblastic phenotype. Cell-cell adhesions maintained by adherens and tight junctions are important for the maintenance of RPE phenotype, and disruption of these junctional complexes results in EMT via activation of signaling pathways such as ß-catenin/Wnt and Hippo signaling, as well as transcription factors involving Zeb1, Snail, and ZONAB. Upon EMT, RPE cells can further differentiate into myofibroblasts in the presence of TGF-ß with cytoskeletal tension mediated by RhoGTPase. These fibroblasts and myofibroblasts derived from RPE cells can contribute to ERM formation by cell migration, proliferation and matrix modification, and play a key role in ERM contraction. It is not solely the proliferation of these cells that results in PVR but rather the contraction of these cells in the ERM.

Role of retinal pigment epithelial cell ß-catenin signaling in experimental proliferative vitreoretinopathy.

Proliferative vitreoretinopathy is caused by the contraction of fibrotic membranes on the epiretinal surface of the neurosensory retina, resulting in a traction retinal detachment and loss of visual acuity. Retinal pigment epithelial (RPE) cells play an important role in formation of such fibrotic, contractile membranes. We investigated the role of Wnt/ß-catenin signaling, a pathway implicated in several fibrotic diseases, in RPE cells in proliferative vitreoretinopathy. In vitro culture of swine RPE sheets resulted in nuclear translocation of ß-catenin in dedifferentiated RPE cells. FH535, a specific inhibitor of ß-catenin signaling, reduced the outgrowth of cultured RPE sheets and prevented dedifferentiated RPE cell proliferation and migration. It also inhibited formation of contractile membranes by dedifferentiated RPE cells on collagen I matrices. Expression and function of the ß-catenin signaling target connexin-43 were down-regulated by FH535, and functional blockade of connexins with carbenoxolone also prevented the in vitro formation of fibrotic, contractile membranes. Intravitreal injection of FH535 in swine also inhibited formation of dense, contractile membranes on the epiretinal surface and prevented development of traction retinal detachment. These findings demonstrate that ß-catenin signaling is involved in formation of contractile membranes by dedifferentiated RPE cells and suggest that adjunctive treatment targeting this pathway could be useful in preventing proliferative vitreoretinopathy.

Dasatinib affects focal adhesion and myosin regulation to inhibit matrix contraction by Müller cells.

Epiretinal membrane (ERM) contraction is associated with a variety of ocular diseases that cause macular dysfunction. Trans-differentiated Müller cells have been identified in ERMs, and have been implicated to be involved in the contractile process. In this study, we tested the effect of dasatinib, an FDA-approved tyrosine kinase inhibitor, on matrix contraction caused by Müller cells, and examined molecular mechanism of action. Type I collagen matrix contraction assays were used to examine the effect of drugs on matrix contraction by trans-differentiated Müller cells. Fluophore-conjugated phalloidin was used for the detection of actin cytoskeleton, and Western-blot analyses were carried out to examine protein expression and phosphorylation status. Dasatinib inhibited collagen matrix contraction by trans-differentiated Müller cells that was associated with decreased cell spreading and reduction of actomyosin stress fibers. Concomitantly, dasatinib-treated Müller cells had reduced phosphorylation of Src family kinase, paxillin, as well as myosin II light chain. Specific inhibitors of Rho/ROCK and myosin II confirmed the critical role played by this pathway in Müller cell contraction. Our data demonstrate that dasatinib significantly reduced matrix contraction by Müller cells via inhibition of focal adhesion, as well as actomyosin contraction.

Therapeutic targeting of telomerase ameliorates experimental choroidal neovascularization.

Choroidal neovascularization (CNV) is the principal driver of blindness in neovascular age-related macular degeneration (nvAMD). Increased activity of telomerase, has been associated with endothelial cell proliferation, survival, migration, and invasion in the context of tumor angiogenesis. Expanding on this knowledge, we investigated the role of telomerase in the development of CNV in mouse model. We observed increased gene expression and activity of telomerase in mouse CNV. Genetic deficiency of the telomerase components, telomerase reverse transcriptase (Tert) and telomerase RNA component (Terc) suppressed laser-induced CNV in mice. Similarly, a small molecule inhibitor of TERT (BIBR 1532), and antisense oligonucleotides (ASOs) targeting Tert and Terc reduced CNV growth. Bone marrow chimera studies suggested that telomerase activity in non-bone marrow-derived cells is crucial for the development of CNV. Comparison of BIBR 1532 with VEGF neutralizing therapeutic strategy in mouse revealed a comparable level of angiosuppressive activity. However, when BIBR and anti-VEGF antibodies were administered as a combination at sub-therapeutic doses, a statistically significant suppression of CNV was observed. These findings underscore the potential benefits of combining sub-therapeutic doses of BIBR and anti-VEGF antibodies for developing newer therapeutic strategies for NV-AMD. Telomerase inhibition with BIBR 1532 suppressed induction of multiple cytokines and growth factors critical for neovascularization. In conclusion, our study identifies telomerase as a promising therapeutic target for treating neovascular disease of the eye and thus provides a proof of principle for further exploration of telomerase inhibition as a novel treatment strategy for nvAMD.

Lens ion transport: from basic concepts to regulation of Na,K-ATPase activity.

In the late 1960s, studies by George Duncan explained many of the basic principles that underlie lens ion homeostasis. The experiments pointed to a permeability barrier close to the surface of the lens and illustrated the requirement for continuous Na,K-ATPase-mediated active sodium extrusion. Without active sodium extrusion, lens sodium and calcium content increases resulting in lens swelling and deterioration of transparency. Later, Duncan's laboratory discovered functional muscarinic and purinergic receptors at the surface of the lens. Recent studies using intact lens suggest purinergic receptors might be involved in short-term regulation of Na,K-ATPase in the epithelium. Purinergic receptor agonists ATP and UTP selectively activate certain Src family tyrosine kinases and stimulate Na,K-ATPase activity. This might represent part of a control mechanism capable of adjusting, perhaps fine tuning, lens ion transport machinery.

Cisplatin-mediated activation of extracellular signal-regulated kinases 1/2 (ERK1/2) by inhibition of ERK1/2 phosphatases.

The mechanism(s) underlying neurodegeneration-associated activation of ERK1/2 remain poorly understood. We report that in cultured rat cortical neurons, whose basal ERK1/2 phosphorylation required NMDA receptors (NMDAR), the neurotoxic DNA intercalating drug cisplatin increased ERK1/2 phosphorylation via NMDAR despite reducing their activity. The rate of ERK1/2 dephosphorylation was lowered by cisplatin. Cisplatin-treated neurons showed general transcription inhibition likely accounting for the reduced expression of the ERK1/2-selective phosphatases including the dual specificity phosphatase-6 (DUSP6) and the DUSP3 activator vaccinia-related kinase-3 (VRK3). Hence, cisplatin effects on ERK1/2 may be due to the deficient ERK1/2 inhibition by the transcription-regulated phosphatases. Indeed, the transcription inhibitor actinomycin D reduced expression of DUSP6 and VRK3 while inducing the NMDAR-dependent activation of ERK1/2 and the impairment of ERK1/2 dephosphorylation. Thus, cisplatin-mediated transcriptional inhibition of ERK1/2 phosphatases contributed to delayed and long lasting accumulation of phospho-ERK1/2 that was driven by the basal NMDAR activity. Our results provide the first direct evidence for transcriptionally-regulated inactivation of neuronal ERK1/2. Its disruption likely contributes to neurodegeneration-associated activation of ERK1/2.

Primary culture of porcine nonpigmented ciliary epithelium.

PURPOSE: Primary culture of nonpigmented ciliary epithelium (NPE) has proved difficult in the past. Here we report development of a method of growing and maintaining primary cultures of NPE from porcine eye. Studies were conducted to confirm that the cultured NPE expressed proteins characteristic of native NPE. METHODS: Intact rings of NPE were isolated from adult pig eyes. A mixture of hyaluronidase and collagenase was used to detach the cells from the basement membrane and vitreous. Dispersed cells were seeded at high density and grown in DMEM with 20% fetal bovine serum under 5% CO2 and 95% air. Protein expression was examined by immunohistochemistry and immunoblot analysis. RESULTS: NPE cells were grown in primary culture and maintained up to 10th passage. Analysis of the ciliary body showed three Na, K-ATPase isoforms (alpha 1, alpha 2, alpha 3) and three nitric oxide synthase isoforms (eNOS, nNOS, iNOS) enriched in the NPE layer but weaker or absent in the PE layer. Each of these proteins as well as the tight junction-specific protein ZO-1 was detected in the cultured NPE. CONCLUSIONS: We developed a simple and reliable way to isolate, culture, and maintain NPE cells from porcine eyes. Success of the method hinged on our ability to isolate pure NPE in large number, detach the cells from the vitreous, and seed the cells at high density. The cultured cells express several proteins that are characteristic of native NPE. NPE cells cultured in this way may prove to be valuable for the study of ciliary body function.

Evaluation of intraretinal migration of retinal pigment epithelial cells in age-related macular degeneration using polarimetric imaging.

The purpose of the present study was to evaluate the intraretinal migration of the retinal pigment epithelium (RPE) cells in age-related macular degeneration (AMD) using polarimetry. We evaluated 155 eyes at various AMD stages. Depolarized light images were computed using a polarization-sensitive scanning laser ophthalmoscope (PS-SLO), and the degree of polarization uniformity was calculated using polarization-sensitive optical coherence tomography (OCT). Each polarimetry image was compared with the corresponding autofluorescence (AF) images at 488 nm (SW-AF) and at 787 nm (NIR-AF). Intraretinal RPE migration was defined by the presence of depolarization at intraretinal hyperreflective foci on PS-SLO and PS-OCT images, and by the presence of hyper-AF on both NIR-AF and SW-AF images. RPE migration was detected in 52 of 155 eyes (33.5%) and was observed in drusenoid pigment epithelial detachment (PED) and serous PED with significantly higher frequencies than in other groups (P = 0.015). The volume of the migrated RPE cluster in serous PED was significantly correlated with the volume of the PED (R2 = 0.26; P = 0.011). Overall, our results showed that intraretinal RPE migrations occurred in various AMD stages, and that they occurred more commonly in eyes with serous and drusenoid PED.

Studies of tyrosine phosphorylation and Src family tyrosine kinases in the lens epithelium.

PURPOSE: Tyrosine phosphorylation regulates many aspects of cell function; thus, cells that have different roles often display different patterns of tyrosine phosphorylation. Because there is interest in differential function of the anterior and equatorial regions of the lens epithelium, studies were conducted to compare tyrosine phosphorylation in the two zones. METHODS: Anterior and equatorial regions of the porcine lens epithelium were collected. Using Western blot analysis, tissue homogenates were probed for tyrosine kinase proteins, phospho-Src, phosphotyrosine, and proliferating cell nuclear antigen (PCNA). RESULTS: Phosphotyrosine immunoblots revealed a marked difference between the pattern of tyrosine phosphorylation in anterior and equatorial regions of the epithelium. Many more bands were detected in the equatorial region, and band density was greater. The abundance of total and active Src family kinases was higher at the equator than at the anterior epithelium. Src kinase activity, which was measured directly by quantifying phosphorylation of a synthetic target peptide using (32)P-gamma-ATP, was detectable only at the equator. In organ-cultured lenses, PP2, a specific inhibitor of the Src kinase family, reduced the density of the phosphotyrosine protein bands. The abundance of PCNA, a protein expressed in proliferating cells, also was reduced in PP2-treated lenses. CONCLUSIONS: The results suggest that the higher Src family kinase activity at the equator contributes to the higher degree of protein phosphorylation observed in this region. The ability of PP2 to suppress PCNA expression suggests a possible link between the activity of Src family kinases and cell proliferation.

Expression, regulation and function of Na,K-ATPase in the lens.

Na,K-ATPase is responsible for maintaining the correct concentrations of sodium and potassium in lens cells. Na,K-ATPase activity is different in the two cell types that make up the lens, epithelial cells and fibers; specific activity in the epithelium is higher than in fibers. In some parts of the fiber mass Na,K-ATPase activity is barely detectable. There is a large body of evidence that suggests Na,K-ATPase-mediated ion transport by the epithelium contributes significantly to the regulation of ionic composition in the entire lens. In some species different Na,K-ATPase isoforms are present in epithelium and fibers but in general, fibers and epithelium express a similar amount of Na,K-ATPase protein. Turnover of Na,K-ATPase by protein synthesis may contribute to preservation of high Na,K-ATPase activity in the epithelium. In ageing lens fibers, oxidation, and glycation may decrease Na,K-ATPase activity. Na,K-ATPase activity in lens fibers and epithelium also may be subject to regulation as the result of protein tyrosine phosphorylation. Moreover, activation of G protein-coupled receptors by agonists such as endothelin-1 elicits changes of Na,K-ATPase activity. The asymmetrical distribution of Na,K-ATPase activity in the epithelium and fibers may contribute to ionic currents that flow in and around the lens. Studies on human cataract and experimental cataract in animals reveal changes of Na,K-ATPase activity but no clear pattern is evident. However, there is a convincing link between abnormal elevation of lens sodium and the opacification of the lens cortex that occurs in age-related human cataract.

Sodium-calcium exchange influences the response to endothelin-1 in lens epithelium.

Studies were conducted to examine the possible involvement of Na+-Ca2+ exchanger in determining the magnitude of the endothelin-1 (ET-1)-receptor-mediated calcium signal in porcine lens epithelial cells. Cytoplasmic calcium concentration was measured in primary cultured cells loaded with Fura-2. ET-1 (100 nM) caused cytoplasmic calcium to increase transiently to approximately 250 nM from a baseline of approximately 65 nM. The calcium increase decayed to a sustained plateau 35-45 nM above the baseline. Both the peak and plateau component of the ET-1 calcium response were abolished by PD145065, an ET receptor antagonist, and by cyclopiazonic acid (CPA) (10 microM). In calcium-free bathing solution, only the plateau was abolished. In the presence of ouabain, low-sodium bathing solution or bepridil, a sodium-calcium exchange inhibitor, peak height more than doubled. Bepridil also increased the peak height of the calcium response to ATP. The half-time for decay of the ET-1 and ATP calcium peak was increased several folds by bepridil, ouabain and low-sodium conditions. Measurements of ionomycin-releasable calcium suggested calcium store size was not increased in bepridil-treated cells. Taken together findings suggest inhibition of sodium-calcium exchange increases the magnitude of the receptor-initiated store-release phase of the ET-1 calcium signaling response as the result of impaired calcium clearance from the cytoplasm.

TGF-beta2-induced matrix modification and cell transdifferentiation in the human lens capsular bag.

PURPOSE: To study the role of TGF-beta2 in posterior capsule opacification (PCO) and to determine whether CAT-152 (lerdelimumab), a fully human monoclonal antibody that neutralizes the effect of TGF-beta2, can also provide therapeutic benefit for PCO. METHODS: In vitro capsular bags were prepared from human donor eyes and maintained in a 5% CO(2) atmosphere at 35 degrees C. To investigate expression of active TGF-beta2, capsular bags were incubated in serum-free EMEM for 2, 28, or more than 100 days and analyzed by ELISA (n > or = 4 at each time point). To study underlying mechanisms, match-pair experiments were also performed, so that the medium was supplemented with 0, 1 or 10 ng/mL TGF-beta2 with or without 10 microg/mL CAT-152 (n = 4 in all cases). On-going observations were by phase-contrast microscopy. In addition, donor material from patients who had undergone cataract surgery was analyzed. Cellular architecture was examined by fluorescence cytochemistry. Expression of matrix metalloproteinase (MMP)-2 and -9 was assessed by gelatin zymography. RESULTS: Analysis of capsular bags from donor eyes that had received an intraocular lens (IOL) revealed the presence of endogenous active TGF-beta2, matrix wrinkling, and expression of transdifferentiation markers alphaSMA and fibronectin. When cultured in vitro, donor bags also showed sustained release of MMP-2 and -9. Culture of capsular bags prepared in vitro from whole lenses showed that TGF-beta2 (1-10 ng/mL) stimulated transdifferentiation and contraction of the capsular bag, resulting in light scatter. TGF-beta2 also induced sustained release of MMP-2 and -9. Active TGF-beta2 was detected in these cultures. The human monoclonal anti-TGF-beta2 antibody CAT-152 (10 microg/mL) effectively inhibited all TGF-beta2-induced effects. CONCLUSIONS: Addition of TGF-beta2 accelerates transdifferentiation and contraction of the capsular bag, resulting in light scatter. CAT-152 inhibited all the effects of TGF-beta2 that were examined and therefore has the potential to suppress development of PCO and provide potential therapeutic benefit to cataract patients.

Regional distribution of Na,K-ATPase activity in porcine lens epithelium.

PURPOSE: It has been established that Na,K-ATPase activity is higher in lens epithelium than fibers. However, others have suggested the Na,K-ATPase enzyme may be inactive or absent in the central 10% of the epithelium. Studies were conducted to measure and compare Na,K-ATPase specific activity and to examine Na,K-ATPase protein expression in the anterior and equatorial regions of porcine lens epithelium. METHODS: Na,K-ATPase activity was determined by measuring the ouabain-sensitive rate of adenosine triphosphate (ATP) hydrolysis. Western blot analysis was used to detect Na,K-ATPase catalytic subunit (alpha) and glycoprotein subunit (beta) protein as well as beta-actin which was used as a loading control. RESULTS: Na,K-ATPase specific activity was more than two times higher in the equatorial epithelium than the anterior 50% of the epithelium. However, the abundance of Na,K-ATPase alpha1 isoform protein was similar in the two regions. Neither the alpha2 nor alpha3 Na,K-ATPase isoform could be detected in the anterior or equatorial epithelium, but Na,K-ATPase beta1 protein was detected in both regions. In contrast to the observed regional difference in Na,K-ATPase activity, the activity of a different P-type ATPase, plasma membrane Ca-ATPase (PMCA), was not significantly different in the anterior and central epithelium. Western blot analysis indicated the presence of two PMCA isoforms, PMCA2, and PMCA4. CONCLUSIONS: Na,K-ATPase activity is significantly higher at the equatorial region of the epithelium compared with the anterior, even though the level of Na,K-ATPase protein is similar in the two regions. It is possible that nonuniform distribution of functional Na,K-ATPase activity contributes to the driving force for circulating solute movement through the lens fiber mass.

Regional differences in tyrosine kinase receptor signaling components determine differential growth patterns in the human lens.

PURPOSE: The lens epithelium can be separated into two regions, the nondividing central zone and the equator, the site of all division in the normal lens. In the present study, the distribution of epithelial growth factor (EGF)/epithelial growth factor receptor (EGFR) signaling components was investigated and related to mitotic distribution in the lens. METHODS: Anterior and equatorial regions of the native epithelium were prepared separately from donor lenses. In vitro capsular bags were prepared from donor eyes and cultured. Receptor distribution was determined by immunocytochemistry and RT-PCR. Western blot analysis of phospholipase C (PLC)-gamma and extracellular signal-regulated kinase (ERK; total and active) was performed on cell lysates. Function was determined by calcium imaging of Fura-2-AM-loaded cells and also, in the case of capsular bags, by cell growth. RESULTS: Immunocytochemistry and RT-PCR showed an even distribution of EGFR across the native epithelium. Whole lenses, however, exhibited only a calcium response to EGF (10 ng/mL) at the equatorial region. Western blot analysis demonstrated significantly greater expression of PLCgamma and ERK (total and active) in the equator than in the central region. Addition of EGF increased growth rates of cells in capsular bags and an EGFR inhibitor decreased rates. EGF also induced a calcium response in posterior capsule cells in the capsular bags. CONCLUSIONS: EGFR is evenly distributed across the entire epithelium, whereas related calcium signaling and expression of PLCgamma and ERK have a marked bias to the equator. Therefore, levels of downstream enzyme components rather than changes in receptor expression dictate EGFR signaling output in the normal lens. In the wounded lens (capsular bag) EGFR signaling persists in cells growing on the posterior capsule.

Inhibition of PVR with a tyrosine kinase inhibitor, dasatinib, in the swine.

PURPOSE: We tested the efficacy of dasatinib, a Food and Drug Administration (FDA)-approved tyrosine kinase inhibitor, to prevent proliferative vitreoretinopathy (PVR). METHODS: The effect of dasatinib on RPE sheet growth was determined by measuring enlargement of cultured RPE sheets in the presence or absence of dasatinib. Epithelial-mesenchymal transition (EMT) of RPE cells was assessed by expression of S100A4. A scratch wound assay, cell number count, and type I collagen contraction assay were used to examine the effect of dasatinib on migration, proliferation, and extracellular matrix (ECM) contraction, respectively. Our swine model of experimental PVR with green fluorescent protein-positive (GFP+) RPE cells was used to assess the efficacy of dasatinib in preventing traction retinal detachment (TRD) caused by PVR. Full-field electroretinography and histologic examination were used to determine the retinal toxicity of dasatinib. RESULTS: Dasatinib prevented RPE sheet growth, cell migration, proliferation, EMT, and ECM contraction in a concentration-dependent manner. 0.1 µM dasatinib inhibited nearly 80% of vitreous fluid-stimulated collagen gel contraction. Dasatinib also prevented TRD caused by PVR in vivo. Only 1/11 eyes had a TRD in the presence of dasatinib, while all 11 controls eyes had a TRD. Dasatinib did not cause any detectable toxicity of the retina. CONCLUSIONS: Dasatinib significantly inhibited PVR-related RPE changes in vitro and prevented TRD in an experimental PVR model in the swine without any detectable toxicity. Our data suggested that dasatinib may be effective in the prevention of PVR.

Proliferative vitreoretinopathy in the Swine-a new model.

PURPOSE: To develop a large animal model of proliferative vitreoretinopathy (PVR) in the swine to eventually study disease pathophysiology, as well as novel therapies. METHODS: PVR was induced in domestic swine by creation of a posterior vitreous detachment, creation of a retinal detachment by the injection of subretinal fluid, and intravitreal injection of green fluorescent protein-positive retinal pigment epithelial (GFP+ RPE) cells. Control eyes had the same surgical procedures without RPE cell injection. PVR was clinically graded on days 3, 7, and 14. Animals were euthanized on day 14, and enucleated eyes were analyzed by light microscopy and immunohistochemistry. RESULTS: Injection of GFP+ RPE cells into the vitreous cavity produced localized, traction retinal detachments by day 14 in all eyes (14 of 14); in contrast, the retina spontaneously reattached by day 3 and remained attached in all control eyes (10 of 10). Contractile epiretinal membranes on the inner retinal surface that caused the traction retinal detachments consisted predominantly of GFP+ RPE cells. These cells stained positive for cytokeratin, confirming their epithelial origin, and also expressed α-SMA and fibronectin, markers for myofibroblasts and fibrosis, respectively. CONCLUSIONS: We established a swine PVR model that recapitulates key clinical features found in humans and, thus, can be used to study the pathophysiology of PVR, as well as new novel therapies. GFP+ RPE cells injected into the vitreous cavity formed contractile membranes on the inner retinal surface and caused localized traction retinal detachments.

Epithelial-mesenchymal transition and proliferation of retinal pigment epithelial cells initiated upon loss of cell-cell contact.

PURPOSE: Molecular mechanisms that initiate epithelial-mesenchymal transition (EMT) involved in ocular fibrotic complications remain elusive. Studies were conducted to examine the role of cell-cell contact in regulating EMT and proliferation of retinal pigment epithelial (RPE) cells. METHODS: Porcine RPE cells were isolated as sheets and cultured in vitro on lens capsules. Cell morphology was examined by microscopy. Western blot analysis and immunostaining were used to follow protein expression. Cell proliferation and RPE function were assessed by BrdU incorporation and phagocytosis assay, respectively. RESULTS: RPE cells in the center of each sheet maintained cell-cell contacts and retained a differentiated phenotype. Disruption of cadherin function in these cells resulted in the loss of cell-cell contact and the concomitant induction of mesenchymal marker protein expression and cell proliferation. RPE cells at the edge of the sheet migrated away from the sheet, underwent EMT, and initiated proliferation, which was accompanied by a switch in cadherin expression from P-cadherin to N-cadherin. Although TGF-beta is thought to be a classic inducer of EMT, it was unable to initiate EMT in RPE cells maintaining cell-cell contact. However, change to alpha-SMA-positive myofibroblasts was induced by TGF-beta in cells that had already undergone EMT. CONCLUSIONS: EMT and the onset of proliferation in RPE cells is initiated by loss of cell-cell contact. TGF-beta cannot initiate EMT or the proliferation of RPE cells maintaining cell-cell contact but appears to play an important secondary role downstream of EMT in inducing transition to a myofibroblast phenotype-a phenotype linked to the development of fibrotic complications.