增生性玻璃体视网膜病变动物模型的改进和评价

增生性玻璃体视网膜病变是导致视网膜脱离手术失败的主要原因,建立动物模型能更好的研究其发病机理,从而进行防治。本文就PVR动物模型的所用动物、模型的建立方法以及评价和分级进行综述。     

METTL3 attenuates proliferative vitreoretinopathy and epithelial-mesenchymal transition of retinal pigment epithelial cells via wnt/β-catenin pathway

Proliferative vitreoretinopathy (PVR) is a refractory vitreoretinal fibrosis disease, and epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is the key pathological mechanism of PVR. However, few studies focused on the role of METTL3, the dominating methyltransferase for m6A RNA modification in PVR pathogenesis. Immunofluorescence staining and qRT-PCR were used to determine the expression of METTL3 in human tissues. Lentiviral transfection was used to stably overexpress and knockdown METTL3 in ARPE-19 cells. MTT assay was employed to study the effects of METTL3 on cell proliferation. The impact of METTL3 on the EMT of ARPE-19 cells was assessed by migratory assay, morphological observation and expression of EMT markers. Intravitreal injection of cells overexpressing METTL3 was used to assess the impact of METTL3 on the establishment of the PVR model. We found that METTL3 expression was less in human PVR membranes than in the normal RPE layers. In ARPE-19 cells, total m6A abundance and the METTL3 expression were down-regulated after EMT. Additionally, METTL3 overexpression inhibited cell proliferation through inducing cell cycle arrest at G0/G1 phase. Furthermore, METTL3 overexpression weakened the capacity of TGFβ1 to trigger EMT by regulating wnt/β -catenin pathway. Oppositely, knockdown of METTL3 facilitated proliferation and EMT of ARPE-19 cells. In vivo, intravitreal injection of METTL3-overexpressing cells delayed the development of PVR compared with injection of control cells. In summary, this study suggested that METTL3 is involved in the PVR process, and METTL3 overexpression inhibits the EMT of ARPE-19 cells in vitro and suppresses the PVR process in vivo.     

Yes-associated protein is essential for proliferative vitreoretinopathy development via the epithelial-mesenchymal transition in retinal pigment epithelial fibrosis

This study was aim to investigate whether the progression of proliferative vitreoretinopathy (PVR) depended on the activation of Yes-associated protein (YAP) and the subsequent epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cell. The effect of YAP activation on retinal fibrosis in a PVR mouse model and in human ARPE-19 cells in vitro was studied. After treated with transforming growth factor-β2(TGF-β2), the expressions of fibrogenic molecules, YAP activation and the TGF-β2-Smad signalling pathway in ARPE-19 cells were detected by Western blot and immunocytochemical analyses. The effect of YAP on change in fibrosis and EMT was tested by knockdown experiment using verteporfin (YAP inhibitor). YAP was upregulated in the PVR mouse model and during TGF-β2–induced RPE cell EMT. In an in vivo study, verteporfin attenuated PVR progression in a mouse model. Additionally, YAP knockdown retained phenotype of RPE cells and ameliorated TGF-β2–induced migration, gel contraction and EMT in vitro. YAP knockdown inhibited the TGF-β2–induced upregulation of connective tissue growth factor (CTGF), smooth muscle actin (SMA-α) and fibronectin. YAP was essential for the TGF-β2–induced nuclear translocation and phosphorylation of Smad2/3. Our work provides direct evidence that YAP is an essential regulator of EMT and profibrotic responses in PVR and indicates that YAP inhibition could be a potential target in PVR therapeutic intervention.     

KRT8 phosphorylation regulates the epithelial-mesenchymal transition in retinal pigment epithelial cells through autophagy modulation

Proliferative vitreoretinopathy (PVR) is a severe ocular disease which results in complex retinal detachment and irreversible vision loss. The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is considered to be critical in the pathogenesis of PVR. In this study, we focused on the potential impact of keratin 8 (KRT8) phosphorylation and autophagy on TGF-β2–induced EMT of RPE cells and explored the relationship between them. Using immunofluorescence and Western blot analysis, the co-localization of KRT8 and autophagy marker, as well as the abundance of phosphorylated KRT8 (p-KRT8) expression, was observed within subretinal and epiretinal membranes from PVR patients. Moreover, during TGF-β2–induced EMT process, we found that p-KRT8 was enhanced in RPE cells, which accompanied by an increase in autophagic flux. Inhibition of autophagy with pharmacological inhibitors or specific siRNAs was associated with a reduction in cell migration and the synthesis of several EMT markers. In the meantime, we demonstrated that p-KRT8 was correlated with the autophagy progression during the EMT of RPE cells. Knockdown the expression or mutagenesis of the critical phosphorylated site of KRT8 would induce autophagy impairment, through affecting the fusion of autophagosomes and lysosomes. Therefore, this study may provide a new insight into the pathogenesis of PVR and suggests the potential therapeutic value of p-KRT8 in the prevention and treatment of PVR.     

Fibrotic Remodeling of the Extracellular Matrix through a Novel (Engineered,Dual-Function) Antibody Reactive to a Cryptic Epitope on the N-Terminal 30 kDa Fragment of Fibronectin

Fibrosis is characterized by excessive accumulation of scar tissue as a result of exaggerated deposition of extracellular matrix (ECM), leading to tissue contraction and impaired function of the organ. Fibronectin (Fn) is an essential component of the ECM, and plays an important role in fibrosis. One such fibrotic pathology is that of proliferative vitreoretinopathy (PVR), a sight-threatening complication which develops as a consequence of failure of surgical repair of retinal detachment. Such patients often require repeated surgeries for retinal re-attachment; therefore, a preventive measure for PVR is of utmost importance. The contractile membranes formed in PVR, are composed of various cell types including the retinal pigment epithelial cells (RPE); fibronectin is an important constituent of the ECM surrounding these cells. Together with the vitreous, fibronectin creates microenvironments in which RPE cells proliferate. We have successfully developed a dual-action, fully human, fibronectin-specific single chain variable fragment antibody (scFv) termed Fn52RGDS, which acts in two ways: i) binds to cryptic sites in fibronectin, and thereby prevents its self polymerization/fibrillogenesis, and ii) interacts with the cell surface receptors, ie., integrins (through an attached “RGD” sequence tag), and thereby blocks the downstream cell signaling events. We demonstrate the ability of this antibody to effectively reduce some of the hallmark features of fibrosis - migration, adhesion, fibronectin polymerization, matrix metalloprotease (MMP) expression, as well as reduction of collagen gel contraction (a model of fibrotic tissue remodeling). The data suggests that the antibody can be used as a rational, novel anti-fibrotic candidate.     

Vascular endothelial growth factor A competitively inhibits platelet-derived growth factor (PDGF)-dependent activation of PDGF receptor and subsequent signaling events and cellular responses

Certain platelet-derived growth factor (PDGF) isoforms are associated with proliferative vitreoretinopathy (PVR), a sight-threatening complication that develops in a subset of patients recovering from retinal reattachment surgery. Although these PDGF isoforms are abundant in the vitreous of patients and experimental animals with PVR, they make only a minor contribution to activating PDGF receptor α (PDGFRα) and driving experimental PVR. Rather, growth factors outside of the PDGF family are the primary (and indirect) agonists of PDGFRα. These observations beg the question of why vitreal PDGFs fail to activate PDGFRα. We report here that vitreous contains an inhibitor of PDGF-dependent activation of PDGFRα and that a major portion of this inhibitory activity is due to vascular endothelial cell growth factor A (VEGF-A). Furthermore, recombinant VEGF-A competitively blocks PDGF-dependent binding and activation of PDGFR, signaling events, and cellular responses. These findings unveil a previously unappreciated relationship between distant members of the PDGF/VEGF family that may contribute to pathogenesis of a blinding eye disease.     

Guidance of cell migration by the Drosophila PDGF/VEGF receptor

Directed cell migrations are important for development, but the signaling pathways and mechanisms responsible for guiding cell migration in vivo are poorly understood. Migration of border cells during Drosophila oogenesis is a simple and attractive model system in which to address these questions. We demonstrate that PVR, a receptor tyrosine kinase related to mammalian PDGF and VEGF receptors, acts in border cells to guide them to the oocyte. The oocyte is the source of a ligand for PVR, PDGF/VEGF factor 1 (PVF1). Intriguingly, the guidance function of PVR is largely redundant with that of EGFR. We present evidence implicating Rac and the Rac activator Mbc/DOCK180/CED-5 as mediators of the guidance signal.     

Rho plays a key role in TGF-beta1-induced cytoskeletal rearrangement in human retinal pigment epithelium

Proliferative vitroretinopathy (PVR) is caused by retinal pigment epithelial (RPE) cell proliferation and transformation into fibrotic cells that produce extracellular matrix (ECM) components. Transforming growth factor beta1 (TGF-beta1) is known to play an important role in PVR pathogenesis. To determine how TGF-beta1 mediates the pathogenic changes in RPE cells, we characterized the effects of TGF-beta1 on the morphology, ECM accumulation, and stress fiber formation of ARPE-19 cells, a human RPE cell line. We then elucidated the signaling pathways that mediate these effects. Serum-starved ARPE-19 cells were incubated with 10 ng/ml TGF-beta1 and their morphological changes were examined by phase-contrast microscopy. Actin reorganization was examined by immunochemistry and confocal microscopy. Protein phosphorylation was analyzed by Western blot analysis. TGF-beta1 treatment induced cytoskeleton reorganization, alpha-SMA expression, increased the phosphorylation of ERK, Smad2/3, and AKT, and activated RhoA and Rac1. Cytoskeletal rearrangement was prevented by pretreatment with a Rho inhibitor and by expression of a dominant negative form of Rho. TGF-beta1 also increased LIM kinase and cofilin phosphorylation and the Rho inhibitor blocked this effect. We propose that TGF-beta1 induces human RPE cells to undergo cytoskeletal actin rearrangement via Rho GTPase-dependent pathways that modulate LIM kinase and cofilin activity. This inhibits actin depolymerization and induces the cytoskeletal rearrangements in RPE cells that result in the characteristic features of PVR.     

Poliovirus entry into human brain microvascular cells requires receptor-induced activation of SHP-2

Viruses use specific receptor molecules to bind selectively to target cells. Receptors have often been considered as mere docking sites, but they may also possess intrinsic signaling capacities that serve to prime the cell for entry and infection. Poliovirus (PV) initiates infection by binding to the PV receptor (PVR) and causes paralytic poliomyelitis by replicating within motor neurons of the brain and spinal cord. We have examined the process by which PV enters cultured human brain microvascular endothelial cells (HBMEC), an in vitro model of the blood-brain barrier. We found that PV enters HBMEC by dynamin-dependent caveolar endocytosis, and that entry depends on intracellular signals triggered by virus attachment to PVR. Tyrosine kinase and RhoA GTPase activation initiated by PVR ligation were both essential. Virus attachment also induced tyrosine phosphorylation of PVR; this permitted the association of PVR with SHP-2, a protein tyrosine phosphatase whose activation was required for entry and infection. The results indicate that receptor-induced signals promote virus entry and suggest a role for tyrosine phosphatases in viral pathogenesis.     

Multiple EGFR ligands participate in guiding migrating border cells

Cell migration is an important feature of embryonic development as well as tumor metastasis. Border cells in the Drosophila ovary have emerged as a useful in vivo model for uncovering the molecular mechanisms that control many aspects of cell migration including guidance. It was previously shown that two receptor tyrosine kinases, epidermal growth factor receptor (EGFR) and PDGF- and VEGF-related receptor (PVR), together contribute to border cell migration. Whereas the ligand for PVR, PVF1, is known to guide border cells, it is unclear which of the four activating EGFR ligands function in this process. We developed an assay to detect the ability of secreted factors to reroute migrating border cells in vivo and tested the activity of EGFR ligands compared to PVF1. Two ligands, Keren and Spitz, guided border cells whereas the other ligands, Gurken and Vein, did not. In addition, only Keren and Spitz were expressed at the appropriate stage in the oocyte, the target of border cell migration. Therefore, a complex combination of EGFR and PVR ligands together guide border cells to the oocyte.     

Programmed Cell Death in Retinal Degeneration: Targeting Apoptosis in Photoreceptors as Potential Therapy for Retinal Degeneration

Retinal degenerations are the major cause of incurable blindness characterized by loss of retinal photoreceptor cells. Several genes causing these genetic diseases have been identified, however the molecular characterization of a high percentage of patients affected by retinitis pigmentosa (RP), a common form of retinal degeneration, is still unknown. The high genetic heterogeneity of these diseases hampers the comprehension of the pathogenetic mechanism causing photoreceptor cell death. Therapies are not available yet and for this reason there is a lot of interest in understanding the etiology and the pathogenesis of these disorders at a cellular and molecular level. Some common features have been identified in different forms of RP. Apoptosis was reported to be the final outcome in all RP animal models and patients analyzed so far. We recently identified two apoptotic pathways co-activated in photoreceptors undergoing cell death in the retinal degeneration (rd1) mouse model of autosomal recessive RP. Our studies opened new perspectives together with many questions that require deeper analyses in order to take advantage of this knowledge and develop new therapeutic approaches. We believe that minimizing cell demise may represent a promising curing strategy that needs to be exploited for retinal degeneration.     

Light-induced photoreceptor apoptosis in vivo requires neuronal nitric-oxide synthase and guanylate cyclase activity and is caspase-3-independent

Apoptosis is the mode of photoreceptor cell death in inherited and induced retinal degeneration. However, the molecular mechanisms of photoreceptor cell death in human cases and animal models of retinal dystrophies remain undefined. Exposure of Balb/c mice to excessive levels of white light results in photoreceptor apoptosis. This study delineates the molecular events occurring during and subsequent to the induction of retinal degeneration by exposure to white light in Balb/c mice. We demonstrate an early increase in intracellular calcium levels during photoreceptor apoptosis, an event that is accompanied by significant superoxide generation and mitochondrial membrane depolarization. Furthermore, we show that inhibition of neuronal nitric-oxide synthase (nNOS) by 7-nitroindazole is sufficient to prevent retinal degeneration implicating a key role for neuronal nitric oxide (NO) in this model. We demonstrate that inhibition of guanylate cyclase, a downstream effector of NO, also prevents photoreceptor apoptosis demonstrating that guanylate cyclase too plays an essential role in this model. Finally, our results demonstrate that caspase-3, frequently considered to be one of the key executioners of apoptosis, is not activated during retinal degeneration. In summary, the data presented here demonstrate that light-induced photoreceptor apoptosis in vivo is mediated by the activation of nNOS and guanylate cyclase and is caspase-3-independent.     

Surface engineering of quantum dots for in vivo vascular imaging

Quantum dot-antibody bioconjugates (QD-mAb) were synthesized incorporating PEG cross-linkers and Fc-shielding mAb fragments to increase in vivo circulation times and targeting efficiency. Microscopy of endothelial cell cultures incubated with QD-mAb directed against cell adhesion molecules (CAMs), when shielded to reduce Fc-mediated interactions, were more specific for their molecular targets. In vitro flow cytometry indicated that surface engineered QD-mAb labeled leukocyte subsets with minimal Fc-mediated binding. Nontargeted QD-mAb nanoparticles with Fc-blockade featured 64% (endothelial cells) and 53% (leukocytes) lower nonspecific binding than non-Fc-blocked nanoparticles. Spectrally distinct QD-mAb targeted to the cell adhesion molecules (CAMs) PECAM-1, ICAM-1, and VCAM-1 on the retinal endothelium in a rat model of diabetes were imaged in vivo using fluorescence angiography. Endogenously labeled circulating and adherent leukocyte subsets were imaged in rat models of diabetes and uveitis using QD-mAb targeted to RP-1 and CD45. Diabetic rats exhibited increased fluorescence in the retinal vasculature from QD bioconjugates to ICAM-1 and VCAM-1 but not PECAM-1. Both animal models exhibited leukocyte rolling and leukostasis in capillaries. Examination of retinal whole mounts prepared after in vivo imaging confirmed the fluorescence patterns seen in vivo. Comparison of the timecourse of retinal fluorescence from Fc-shielded and non-Fc-shielded bioconjugates indicated nonspecific uptake and increased clearance of the non-Fc-shielded QD-mAb. This combination of QD surface design elements offers a promising new in vivo approach to specifically label vascular cells and biomolecules of interest.     

Emerging model systems and treatment approaches for Leber's hereditary optic neuropathy: Challenges and opportunities

Leber's hereditary optic neuropathy (LHON) is a mitochondrial disease mainly affecting retinal ganglion cells (RGCs). The pathogenesis of LHON remains ill-characterized due to a historic lack of effective disease models. Promising models have recently begun to emerge; however, less effective models remain popular. Many such models represent LHON using non-neuronal cells or assume that mutant mtDNA alone is sufficient to model the disease. This is problematic because context-specific factors play a significant role in LHON pathogenesis, as the mtDNA mutation itself is necessary but not sufficient to cause LHON. Effective models of LHON should be capable of demonstrating processes that distinguish healthy carrier cells from diseased cells. In light of these considerations, we review the pathophysiology of LHON as it relates to old, new and future models. We further discuss treatments for LHON and unanswered questions that might be explored using these new model systems.     

Galectin-3 Induces Clustering of CD147 and Integrin-β1 Transmembrane Glycoprotein Receptors on the RPE Cell Surface

Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and integrin-β1 via interaction with β1,6-branched N-glycans on RPE cells and hypothesize that Gal-3 acts as a positive regulator for CD147/integrin-β1 clustering and therefore modifies RPE cell behavior contributing to the pathogenesis of PVR. Further investigations at this pathway may aid in the development of specific therapies for PVR.     

Elevated levels of substance P in intraocular fluid in proliferative vitreoretinopathy.

Proliferative vitreoretinopathy is the most common reason for failure in retinal reattachment surgery. Since both substance P (SP) and SP receptors were found to be present in the human eye, and as pharmacological studies suggest an importance of SP for ocular functions, we investigated intraocular fluids for the presence of SP in eyes elected for cataract surgery, retinal detachment surgery and retina surgery for severe proliferative vitreoretinopathy (PVR) as well as in eyes with proliferative diabetic retinopathy (PDR). High performance liquid chromatography and radioimmunoassay (RIA) for SP immunoreactivities were performed. The SP mean concentration in intraocular fluid (IOF) of patients for cataract surgery (CS) was 2.2 fmol/ml, for retinal detachment (RD) was 2.7 fmol/ml and for PDR was 1.9 fmol/ml; significantly higher levels (mean concentration of 26.9 fmol/ml) were measured in eyes with PVR. HPLC analysis revealed two immunoreactive peaks coeluting with synthetic SP and SP-sulfoxide, indicating that RIA values represent authentic SP. We conclude that SP may play an important role in PVR. Since SP antagonists are known to inhibit a variety of SP effects in the eye, there might be a useful tool to reveal the importance of SP in this disease and, in this instance, a new possible treatment.     

Trichostatin A,a histone deacetylase inhibitor,suppresses proliferation and epithelial–mesenchymal transition in retinal pigment epithelium cells

The proliferation and epithelial–mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells are the major pathological changes in development of proliferative vitreoretinopathy (PVR), which leads to severe visual impairment. Histone deacetylases (HDACs)‐mediated epigenetic mechanisms play important roles in controlling various physiological and pathological events. However, whether HDACs are involved in the regulation of proliferation and EMT in PRE cells remains unidentified. In this study, we evaluated the expression profile of HDAC family (18 genes) and found that some of class I and class II HDACs were up‐regulated in transforming growth factor‐β2 (TGF‐β2)/TGF‐β1‐stimulated RPE cells. Tricostatin A (TSA), a class I and II HDAC inhibitor, suppressed the proliferation of RPE cells by G1 phase cell cycle arrest through inhibition of cyclin/CDK/p‐Rb and induction of p21 and p27. In the meantime, TSA strongly prevented TGF‐β2–induced morphological changes and the up‐regulation of α‐SMA, collagen type I, collagen type IV, fibronectin, Snail and Slug. We also demonstrated that TSA affected not only the canonical Smad signalling pathway but also the non‐canonical TGF‐β/Akt, MAPK and ERK1/2 pathways. Finally, we found that the underlying mechanism of TSA affects EMT in RPE cells also through down‐regulating the Jagged/Notch signalling pathway. Therefore, this study may provide a new insight into the pathogenesis of PVR, and suggests that epigenetic treatment with HDAC inhibitors may have therapeutic value in the prevention and treatment of PVR.