Normal vitreous promotes angiogenesis via activation of Axl.

Vitreous has been reported to prevent tumor angiogenesis, but our previous findings indicate that vitreous activate the signaling pathway of phosphoinositide 3-kinase (PI3K)/Akt, which plays a critical role in angiogenesis. The goal of this research is to determine which role of vitreous plays in angiogenesis-related cellular responses in vitro. We found that in human retinal microvascular endothelial cells (HRECs) vitreous activates a number of receptor tyrosine kinases including Anexelekto (Axl), which plays an important role in angiogenesis. Subsequently, we discovered that depletion of Axl using CRISPR/Cas9 and an Axl-specific inhibitor R428 suppress vitreous-induced Akt activation and cell proliferation, migration, and tuber formation of HRECs. Therefore, this line of research not only demonstrate that vitreous promotes angiogenesis in vitro, but also reveal that Axl is one of receptor tyrosine kinases to mediate vitreous-induced angiogenesis in vitro, thereby providing a molecular basis for removal of vitreous as cleanly as possible when vitrectomy is performed in treating patients with proliferative diabetic retinopathy.

Idelalisib inhibits vitreous-induced Akt activation and proliferation of retinal pigment epithelial cells from epiretinal membranes.

Proliferative vitreoretinopathy (PVR) is a blinding fibrotic eye disease that develops in 8-10% of patients who undergo primary retinal detachment-reparative surgery and in 40-60% of patients with open-globe injury. At present, there is no pharmacological treatment for this devastating disease. Vitreal growth factors activate their respective receptors of cells in the vitreous, trigger their downstream signaling transduction (e.g. phosphoinositide 3 kinases (PI3Ks)/Akt), and drive cellular responses intrinsic to the pathogenesis of PVR. PI3Ks play a central role in experimental PVR. However, which isoform(s) are involved in PVR pathogenesis remain unknown. Herein, we show that p110δ, a catalytic subunit of receptor-regulated PI3K isoform δ, is highly expressed in epiretinal membranes from patients with PVR, and that idelalisib, a specific inhibitor of PI3Kδ, effectively inhibits vitreous-induced Akt activation, proliferation, migration and contraction of retinal pigment epithelial cells derived from an epiretinal membrane of a PVR patient. Small molecules of kinase inhibitors have shown great promise as a class of therapeutics for a variety of human diseases. The data herein suggest that idelalisib is a promising PVR prophylactic.

PKC-ζ Regulates Thrombin-Induced Proliferation of Human Müller Glial Cells.

PURPOSE: To investigate the effect of thrombin on the proliferation of human Müller glial cells (MCs) and define the possible signaling mechanisms involved in this process. METHODS: Protease-activated receptor (PARs 1-4) expression was analyzed using RT-PCR and Western blot in the MIO-M1 Müller cell line (MC). Müller cell proliferation was assessed by the MTS reduction method. Wound healing and immunoreactivity to Ki67 antigen were used to dissociate proliferation and migration. Cell migration was examined using transwell migration assays. The involvement of extracellular signal-regulated kinase (ERK1/2) phosphorylation/activation in thrombin-induced human MC proliferation was determined by Western blot. Intracellular pathways involved in ERK1/2 activation were analyzed by pharmacologic inhibition. RESULTS: We first demonstrated that human MCs express PARs 1 to 4. Our results show that thrombin dose-dependently stimulates MC proliferation by 44%, with a calculated Ec50 of 0.86 nM. Müller cell maximal proliferation required sustained thrombin treatment for 72 hours, in contrast to our previous findings in RPE cells showing maximal thrombin-induced proliferation at 24-hour stimulation. We demonstrate that thrombin induces MC cell proliferation through the Ras-independent activation of the Raf/MEK/ERK cascade, under the control of protein kinase C (PKC)-ζ. CONCLUSIONS: The breakdown of blood-retina barrier (BRB) exposes MCs to thrombin contained in serum. Our findings further strengthen the critical involvement of thrombin in the development of proliferative retinopathies and may provide pharmacologic targets for the prevention or treatment of these diseases.

The Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)-created MDM2 T309G Mutation Enhances Vitreous-induced Expression of MDM2 and Proliferation and Survival of Cells.

The G309 allele of SNPs in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but whether SNP G309 contributes to the pathogenesis of PVR is to date unknown. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) can be harnessed to manipulate a single or multiple nucleotides in mammalian cells. Here we delivered SpCas9 and guide RNAs using dual adeno-associated virus-derived vectors to target the MDM2 genomic locus together with a homologous repair template for creating the mutation of MDM2 T309G in human primary retinal pigment epithelial (hPRPE) cells whose genotype is MDM2 T309T. The next-generation sequencing results indicated that there was 42.51% MDM2 G309 in the edited hPRPE cells using adeno-associated viral CRISPR/Cas9. Our data showed that vitreous induced an increase in MDM2 and subsequent attenuation of p53 expression in MDM2 T309G hPRPE cells. Furthermore, our experimental results demonstrated that MDM2 T309G in hPRPE cells enhanced vitreous-induced cell proliferation and survival, suggesting that this SNP contributes to the pathogenesis of PVR.

TNF-α promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling.

Tumor necrosis factor-alpha (TNF-α) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-α promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-α-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-α-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-α promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

Interleukin-6 and matrix metalloproteinase expression in the subretinal fluid during proliferative vitreoretinopathy: correlation with extent, duration of RRD and PVR grade.

PURPOSE: To investigate interleukin (IL)-6 protein levels in the subretinal fluid (SRF) of patients with rhegmatogenous retinal detachment (RRD) complicated by proliferative vitreoretinopathy (PVR); to correlate the IL-6 levels with matrix metalloproteinases (MMP)-1, -2, -3, -8, -9 and tissue inhibitor of metalloproteinases (TIMP)-1 with respect to RRD extent, duration and PVR grade. METHODS: Thirty-one SRF samples from 31 eyes of 31 patients with RRD complicated with PVR and five SRF samples from five eyes of five patients suffering from RRD not complicated with PVR were collected during treatment by scleral buckling. Enzyme-Linked Immunosorbent Assay was employed for the measurement of IL-6, MMP-1, -3, -8 and TIMP-1 levels while the enzymatic activity of MMP-2 and MMP-9 was assessed by gelatin zymography. RESULTS: Protein levels of IL-6 (p=0.050), MMP-1 (p=0.001), MMP-3 (p=0.005), MMP-8 (p=0.003), TIMP-1 (p=0.001) as well as enzymatic activity of proMMP-2 (p=0.001), MMP-2 (p=0.023) and MMP-9 (p=0.015), were significantly higher in the SRF of PVR patients compared to controls. IL-6 levels correlated significantly with TIMP-1 (r=0.528, p=0.035). Regarding clinical parameters of the detachment, IL-6 levels correlated with RRD extent (r=0.592, p=0.016), but not with RRD duration (p=0.857) and PVR grade (p=0.594). Regression analysis revealed positive correlations between IL-6 and MMP-2. CONCLUSIONS: There was a significant correlation between IL-6 and TIMP-1 levels in the SRF of PVR patients. The findings of this study are in agreement with relevant studies concerning IL-6 involvement in the modulation of MMP expression and are indicative of IL-6 and MMP activity during PVR, mainly that of MMP-2 and TIMP-1.

Vitreous-induced cytoskeletal rearrangements via the Rac1 GTPase-dependent signaling pathway in human retinal pigment epithelial cells.

Proliferative vitreoretinopathy (PVR) is mainly caused by retinal pigment epithelial (RPE) cell migration, invasion, proliferation and transformation into fibroblast-like cells that produce the extracellular matrix (ECM). The vitreous humor is known to play an important role in PVR. An epithelial-to-mesenchymal transdifferentiation (EMT) of human RPE cells induced by 25% vitreous treatment has been linked to stimulation of the mesenchymal phenotype, migration and invasion. Here, we characterized the effects of the vitreous on the cell morphology and cytoskeleton in human RPE cells. The signaling pathway that mediates these effects was investigated. Serum-starved RPE cells were incubated with 25% vitreous, and the morphological changes were examined by phase-contrast microscopy. Filamentous actin (F-actin) was examined by immunofluorescence and confocal microscopy. Protein phosphorylation of AKT, ERK1/2, Smad2/3, LIM kinase (LIMK) 1 and cofilin was analyzed by Western blot analysis. Vitreous treatment induced cytoskeletal rearrangements, activated Rac1 and enhanced the phosphorylation of AKT, ERK1/2 and Smad2/3. When the cells were treated with a Rac activation-specific inhibitor, the cytoskeletal rearrangements were prevented, and the phosphorylation of Smad2/3 was blocked. Vitreous treatment also enhanced the phosphorylation of LIMK1 and cofilin and the Rac inhibitor blocked this effect. We propose that vitreous-transformed human RPE cells undergo cytoskeletal rearrangements via Rac1 GTPase-dependent pathways that modulate LIMK1 and cofilin activity. The TGFß-like activity of the vitreous may participate in this effect. Actin polymerization causes the cytoskeletal rearrangements that lead to the plasticity of vitreous-transformed RPE cells in PVR.

Expression of autotaxin and acylglycerol kinase in proliferative vitreoretinal epiretinal membranes.

PURPOSE: Lysophosphatidic acid (LPA)/LPA(1) receptor pathway is involved in inflammation, angiogenesis and fibrosis. This study was conducted to analyse the expression of LPA-producing enzymes, autotaxin (ATX) and acylglycerol kinase (AGK) and LPA(1) receptor, in proliferative diabetic retinopathy (PDR) and proliferative vitreoretinopathy (PVR) epiretinal membranes. METHODS: Nine active and 13 inactive membranes from patients with PDR and 21 membranes from patients with PVR were studied by immunohistochemistry. RESULTS: In PDR membranes, vascular endothelial cells expressed ATX and AGK in 16 and 19 membranes, respectively. Stromal cells expressed ATX and AGK in 19 and 22 membranes, respectively. Immunoreactivity for LPA(1) receptor was noted in vascular endothelial cells and stromal cells in the five membranes stained for LPA(1) receptor. Numbers of blood vessels and stromal cells expressing CD34, ATX and AGK were significantly higher in active membranes than in inactive membranes. Significant correlations were detected between number of blood vessels expressing the panendothelial cell marker CD34 and number of blood vessels and stromal cells expressing ATX and AGK. In PVR membranes, spindle-shaped myofibroblasts expressing α-smooth muscle actin co-expressed ATX, AGK and LPA(1) receptor. CONCLUSIONS: The LPA/LPA(1) receptor pathway may be involved in inflammatory, angiogenic and fibrotic responses in proliferative vitreoretinal disorders.

Interleukin-6 and the matrix metalloproteinase response in the vitreous during proliferative vitreoretinopathy.

PURPOSE: To investigate the levels of IL-6 in the vitreous of patients with RRD complicated with PVR and correlate the IL-6 levels with matrix metalloproteinase (MMP)-1,-2,-3,-8,-9 and tissue inhibitor of metalloproteinases (TIMP)-1 with respect to RRD extent, duration and PVR grade. DESIGN: Cohort study. PARTICIPANTS: Twenty-eight vitreous samples from 28 eyes of 28 patients with RRD complicated with PVR. METHODS: Institutional study. Twenty-eight vitreous samples from 28 eyes of 28 patients with RRD complicated with PVR were collected during pars plana vitrectomy (PPV) and were compared to vitreous control samples. IL-6, MMP-1,-3,-8 and TIMP-1 levels were measured using ELISA while enzymatic activity of MMP-2, and -9 was determined employing gelatin zymography. RESULTS: Protein IL-6 (p=0.030), MMP-1 (p=0.003), MMP-3 (p=0.003), TIMP-1 (p=0.001) levels as well as enzymatic activity of proMMP-9 (p=0.013), MMP-9 (p=0.017) and proMMP-2 (p=0.010), were significantly increased in PVR patients as compared to controls. IL-6 levels correlated with MMP-1 (p=0.002), proMMP-2 (p=0.006), MMP-3 (p=0.001) and TIMP-1 (p=0.006). Regression analysis revealed positive correlations between IL-6 and all MMPs and TIMP-1. CONCLUSIONS: Taking into account the previously established effect of interleukins in MMP activity, the findings of this study suggest a role of IL-6 in MMP stimulation during PVR development.

Correlation of matrix metalloproteinase levels with the grade of proliferative vitreoretinopathy in the subretinal fluid and vitreous during rhegmatogenous retinal detachment.

PURPOSE: We investigated the activity of matrix metalloproteinase (MMP)-2 and -9 and their latent pro-forms (proMMP-2, -9), and protein levels of MMP-1, -3, -8 and tissue inhibitor of MMPs (TIMP)-1 in the subretinal fluid (SRF) and vitreous of patients with rhegmatogenous retinal detachment (RRD). Potential correlations with proliferative vitreoretinopathy (PVR) grade were determined. METHODS: Thirty-seven SRF and 32 vitreous samples from RRD patients and nine vitreous samples from human organ donors (controls), were collected and assayed for MMP-1, -3, -8/TIMP-1 levels using enzyme-linked immunosorbent assay (ELISA), and for proMMP-2, -9, MMP-2, -9 activity employing gelatine zymography. RESULTS: ProMMP-2, -9, MMP-1, -3, -9, TIMP-1 were significantly higher in the SRF and vitreous of RRD patients compared to the vitreous of organ donors. MMP-8 levels were higher in RRD patients' SRF. Regarding PVR grade, MMPs and TIMP-1 were differentially present in SRF and vitreous. PVR grade correlated significantly with the levels of MMP-2 in SRF, while proMMP-2, MMP-1, -2, -3, -8, -9 and TIMP-1 levels correlated with PVR grade in the vitreous. CONCLUSION: MMP/TIMP-1 levels are elevated in SRF and vitreous during RRD. Significant correlations between PVR grade and MMP-2 in SRF and proMMP-2, MMP-1, -2, -3, -8, -9 and TIMP-1 levels in vitreous were revealed. Investigation of MMP activity in vitreous may provide more valid conclusions compared to SRF pertaining to the role of the MMPs during RRD. The observations of the present study suggest a possible role for MMPs and TIMP-1 in PVR pathophysiology.

Calcium-independent phospholipase A2 regulates retinal pigment epithelium proliferation and may be important in the pathogenesis of retinal diseases.

Calcium-independent phospholipase A2, group VIA (iPLA2-VIA) is involved in cell proliferation. This study aimed to evaluate the role of iPLA2-VIA in retinal pigment epithelium (RPE) cell proliferation and in retinal diseases involving RPE proliferation. A human RPE cell line (ARPE-19) was used to explore this role in vitro. Proliferating ARPE-19 cells had increased expression and activity of iPLA2-VIA. iPLA2-VIA was found in the nuclei of proliferating ARPE-19 cells, whereas in confluent ARPE-19 cells, with limited proliferation, iPLA2-VIA was primarily found in the cytosol. Inhibition of iPLA2-VIA decreased the rate of proliferation, whereas over expression of iPLA2-VIA increased the rate of proliferation. Using an experimental porcine model of RPE proliferation we demonstrated significant nuclear upregulation of iPLA2-VIA in proliferating RPE cells in vivo. We furthermore evaluated the expression of iPLA2-VIA in proliferative vitreoretinopathy (PVR). PVR membranes revealed nuclear expression of iPLA2-VIA in the RPE cells which had migrated and participated in the formation of the membranes. Overall, the present results point to an important role of iPLA2-VIA in the regulation of RPE proliferation suggesting that iPLA2-VIA may be considered as a possible pharmaceutical target in retinal diseases involving RPE proliferation and migration.

Targeting of integrin-linked kinase with a small interfering RNA suppresses progression of experimental proliferative vitreoretinopathy.

Integrin-linked kinase (ILK) is a serine/threonine kinase that interacts through its COOH terminus with beta1 and beta3 integrins, which mediates a diversity of cell functions by coupling integrins and growth factors to cascades of downstream signaling events. The purpose of this work was to investigate the effects of ILK on development of experimental proliferative vitreoretinopathy (PVR). Cultured human RPE cell line D407 was knocked down for ILK using a small interfering RNA (siRNA). For this, cellular ILK expression was quantified by real-time quantitative PCR, Western blot analysis and immunocytochemical assay, and cytotoxicity of transfection was determined by MTT assay. Moreover, cell attachment, spreading, migration, microfilament dynamics, and cell cycling assays were performed. Furthermore, the impact of the ILK-specific siRNA on PVR was tested using a rabbit model in which PVR was induced by the injection of human RPE cells. Prevalence of PVR and retinal detachment were determined by indirect ophthalmoscopy on days 1, 3, 7, 14, 21 and 28 post-injection. The results showed that blocking the expression of ILK by siRNA significantly inhibited human RPE cell attachment, spreading, migration and proliferation. The knockdown of ILK also disturbed F-actin assembly and induced a cellular arrest in the G1 phase of the cell cycle. Though the eyes injected with ILK-specific siRNA also developed features of PVR, the severities of day 28 post-injection were significantly lower than those in the control eyes (P<0.01). We conclude that targeting of ILK with a small interfering RNA not only inhibits human RPE cell attachment, spreading, migration and proliferation in vitro, but also effectively suppresses development of proliferative vitreoretinopathy in a rabbit model. This may be a potential therapeutic usefulness in treating PVR.

Lysyl oxidase activity in the ocular tissues and the role of LOX in proliferative diabetic retinopathy and rhegmatogenous retinal detachment.

PURPOSE: Lysyl oxidase (LOX) cross-links the side chain of collagen and elastin and thereby contributes to extracellular matrix (ECM) integrity. ECM remodeling is seen in various ocular diseases. Until now, there have been no reports on the LOX enzyme's activity in ocular tissues. The purpose of this study was to estimate LOX activity and expression in human donor ocular tissues and to measure the specific activity of LOX in the vitreous of proliferative diabetic retinopathy (PDR) and rhegmatogenous retinal detachment (RRD). METHOD: Human donor eyeballs obtained from an eye bank were used to study tissue distribution of LOX. Human vitreous specimens were obtained during vitreoretinal surgery from PDR (n = 16) and RRD (n = 10). LOX activity was estimated by N-acetyl-3,7-dihydroxyphenoxazine assay, immunohistochemistry, and real-time polymerase chain reaction (RT-PCR). Matrix metalloprotease (MMP)-2 and -9 were quantified in the vitreous by sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: The specific activity of LOX in ocular tissues was on the order of vitreous, iris ciliary body, lens, choroid RPE, and retina, which were comparable by mRNA expression and immunolocalization. The vitreous level of LOX activity decreased significantly in PDR and RRD, with an increase in total MMP-2 and -9 levels compared with normal donor vitreous. CONCLUSIONS: LOX activity showed a statistically significant decrease in the vitreous of PDR and RRD relative to control specimens. This effect can contribute to the inadequate collagen cross-linking that causes the ECM changes that occur in these diseases.

The activation of MEK-ERK1/2 by glutamate receptor-stimulation is involved in the regulation of RPE proliferation and morphologic transformation.

Retinal pigment epithelial (RPE) cells are the main cell type involved in the pathogenesis of proliferative vitreoretinopathy (PVR). As a result from retinal detachment or surgical procedures, RPE comes in contact with glutamate from serum, glial release and the injured retina. The purpose of this study was to explore a possible role for glutamate in the development of PVR, mediated by the receptor-stimulated activation of the ERK1/2 MAPK pathway, the alteration of cell proliferation and the transdifferentiation of RPE cells, using rat RPE cells in culture as a model system. We demonstrated the expression in these cells of Group I metabotropic-and ionotropic AMPA/KA and NMDA glutamate receptors (GluRs), predominantly of the NMDA subtype, which are targeted to the membrane, and exhibit pharmacological and biochemical characteristics equivalent to those previously established in brain tissue. Proliferation was measured by MTS-reduction colorimetric assay, and actin cytoskeleton dynamics was visualized by immunoflurescence using alpha-sma specific antibodies. Activation of metabotropic, AMPA and NMDA receptors by glutamate induced the time-and dose-dependent phosphorylation of ERK1/2, assessed by Western blot analysis, in parallel to a significant increase in cell proliferation and a decrease in alpha-sma expression and its recruitment into stress fibers. These effects were all prevented by the inhibition of MEK. Hence, results suggest that glutamate could be involved in the generation of PVR, through a GluR-mediated increase in proliferation and phenotypic transformation, cause-effect related to the activation of ERK1/2.

Expression of matrix metalloproteinases in the subretinal fluid correlates with the extent of rhegmatogenous retinal detachment.

BACKGROUND: We investigated the activity of matrix metalloproteinase (MMP)-2 and -9 and the protein levels of MMP-1, -3, -8 and the tissue inhibitor of MMPs (TIMP)-1 in the subretinal fluid (SRF) of patients with rhegmatogenous retinal detachment (RRD) and establishment of potential correlations with clinical parameters. METHODS: Thirty-seven SRF from RRD patients and nine vitreous samples from the human eye of organ donors (controls) were collected and assayed for MMP-1,-3,-8 and TIMP-1 levels using ELISA and for MMP-2 and -9 activity employing gelatin zymography. RESULTS: MMP-1, MMP-3, MMP-8, proMMP-2, proMMP-9, MMP-9 and TIMP-1 levels were higher in SRF compared with vitreous fluid. Overall, MMPs and TIMPs were differentially expressed in SRF with respect to duration and extent of RRD, as well as to stage of proliferative vitreoretinopathy. Regression analysis for all data indicated that a model consisting of MMP-3, MMP-8 and proMMP-9 could estimate the extent of RRD. CONCLUSIONS: MMPs and TIMP-1 levels are elevated in SRF during RRD. A regression model consisting of MMP-3, MMP-8 and proMMP-9 may be proved to be of potential use in providing information for evaluation of the extent of RRD.

[Changes of protein kinase C for the proliferation of retinal pigment epithelium cells induced by subretinal fluid].

OBJECTIVE: To study the effects of subretinal fluid (SRF) on the proliferation of retinal pigment epithelium (RPE) cells and on the activation and translocation of protein kinase C (PKC); to investigate the relationship between the proliferation and the changes of PKC in the RPE cells; and to study the effect of PKC inhibitor. METHODS: RPE cells were harvested to measure the PKC activity in cytoplasm and cellular membrane after being treated with subretinal fluid (SRF) obtained from PVR patients with different degrees (B grade and C), PKC specific activator PMA (positive control) or normal vitreous. RPE cells cultured with DMEM culture medium only were used as the negative controls. PKC activity in cytoplasm and cellular membrane was measured with radioactive isotope (32)P label method. For further study, the PKC specific inhibitor N, N-dimethyl was used to pretreat the RPE cells before the administration of SRF, PMA or normal vitreous, and then the activity of PKC was observed and recorded. (3)H-TdR was used to measure the proliferation of RPE cells with or without the activation and translocation. RESULTS: SRF and PMA could promote the proliferation of RPE cells and activate PKC in the cytoplasm of RPE cells, and then promoted the PKC translocated from the cytoplasm to cellular membrane. The peak of PRC on the cell membrane appeared later in cells treated with SRF when compared with those treated with PMA. The appearance of peak of PKC in cells treated with SRF from grade B PVR was later than those treated with SRF from grade C PVR, the stimulating effect on the proliferation of RPE cells by the SRF B was also less than those from the SRF C. No activation of PKC and increased proliferation were observed in RPE cells treated with normal vitreous or DMEM culture medium. Pre-treatment with PKC inhibitor could block the PKC-activating effects and proliferation-stimulating effects of SRF and PMA on the RPE cells, therefore no significant difference could be detected between different groups. CONCLUSION: SRF can promote the proliferation of RPE cells and induce the activation and translocation of PKC in RPE cells. PKC is involved in the process of the proliferation of RPE cells. The PKC inhibitor could block this process.

Keratinocyte transglutaminase in proliferative vitreoretinopathy.

PURPOSE: Proliferative vitreoretinopathy (PVR) is an excessive wound-healing process and the major complication in rhegmatogenous retinal detachment. The present study was designed to investigate the expression of keratinocyte transglutaminase (kTgase) in PVR membranes and retinal pigment epithelial (RPE) cells and to evaluate its expression in response to growth factors known to be increased in PVR disease. METHODS: Distribution of kTgase and its relation to fibronectin have been investigated immunohistochemically. PVR membranes and native and cultured RPE cells were analyzed by RT-PCR for the presence of kTgase mRNA. In vitro, RPE cells were treated with transforming growth factor (TGF)-beta2, basic fibroblast growth factor, interleukin-6, and interleukin-1beta. Expression of kTgase was studied by Northern and Western blot analysis. The effect of connective tissue growth factor (CTGF) silencing on the TGF-beta2-modulated expression of kTgase was investigated by transfection with CTGF small interfering (si)RNA before TGF-beta2 treatment. RESULTS: mRNA expression of kTgase was present in all PVR membranes. Immunohistochemical staining for kTgase showed an inhomogeneous pattern with localized accumulation and little colocalization with fibronectin. Although native RPE cells contained only a basal level of kTgase mRNA, the expression of kTgase was increased under culture conditions and was further enhanced by TGF-beta2 treatment. Silencing of CTGF expression did not attenuate the TGF-beta2-mediated induction of kTgase. CONCLUSIONS: The findings demonstrate that kTgase is present in PVR membranes. Its amount is related to the differentiation state of RPE cells and stimulation by TGF-beta2. TGF-beta2-mediated increase seems to be independent of CTGF.

Mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) pathways differently regulate retinal pigment epithelial cell-mediated collagen gel contraction.

Retinal pigment epithelial (RPE) cell-mediated extracellular matrix contraction is believed to contribute to developing proliferative vitreoretinopathy. It has been shown that platelet-derived growth factor (PDGF) and its intracellular signaling pathway, including mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K), are mainly involved in this process. The aim of this study is to investigate how these downstream signaling pathways are related to RPE-mediated collagen gel contraction. We performed the gel contraction assay to evaluate the effect of PDGF in cultured ARPE-19 cells under the presence or absence of PD98059, MAPK inhibitor or wortmannin, PI3K inhibitor. Experiments treated with neutralizing antibody for various subtypes of integrin were also performed and the effect on PDGF-induced gel contraction was investigated. Expression changes of integrin alpha1, alpha2 and beta1 after PDGF stimulation was evaluated using quantitative real-time PCR and flow cytometry. The results showed that PDGF up-regulated ARPE-19 cell-mediated gel contractile activity. PDGF-induced collagen gel contraction was attenuated under presence of PD98059, wortmannin, or neutralizing antibody for integrin alpha1, alpha2, or beta1, all of which are critical subset for binding with type I collagen. The expression of integrin alpha1 and alpha2 was increased after PDGF stimulation in both real-time PCR and flow cytometry, however beta1 expression was not increased. PD98059 significantly attenuated integrin alpha1 and alpha2 expressions. However, wortmannin did not have the same effect. In conclusion, PDGF promotes ARPE-19 cell-mediated gel contraction via both MAPK and PI3K. This was probably due to an increased expression of integrin alpha1 and alpha2, which is mediated by MAPK, but not by PI3K. PI3K may regulate collagen gel contraction by another mechanism other than the up-regulation of integrin expression.

[The role of specific ROCK inhibitor in the prevention of experimental PVR in rabbits].

OBJECTIVE: To determine the effect of Y27632, a specific inhibitor of p160 Rho-associated coiled-coil forming protein kinase (ROCK), on experimental rabbit PVR. METHODS: Cultured rabbit retinal pigment epithelial cells were used in the experiments. The effects of Y27632 on RPE alpha-SMA (smooth muscle actin) stress fiber formation were studied by immuno-fluorescent staining. An in vitro type I collagen gel contraction assay and MTT assay were used to detect the effect of Y27632 on RPE cell contractile force and proliferation. Cultured 6 th passage rabbit RPE cells were injected intravitreally to induce the PVR model and then followed injection of 50 micromol/L of Y27632. The presence of tractional retinal detachment (TRD) was assessed to evaluate the effect of this agent in vivo. Electroretinography and histological studies were performed after intravitreal injection of Y27632 into untreated eyes to evaluate toxicity. RESULTS: The results showed that Y27632 disrupted SMA stress fiber formation in the cultured RPE cells and impaired contractile force generated by RPE cells (t = 16.212, P < 0.01). Development of TRD was significantly reduced (P < 0.01) with 50 micromol/L of Y27632. No obvious histological or retinal functional damage was found in the Y27632-treated group. CONCLUSION: p160 ROCK specific inhibitor Y27632 decreased contractile force generated by RPE cells and attenuated PVR without significant side effect in rabbit. This reagent could be potential therapeutically method in the treatment of PVR.

Involvement of rho-kinase pathway in contractile activity of rabbit RPE cells in vivo and in vitro.

PURPOSE: Increased alpha-smooth muscle actin (alpha-SMA) expression in epiretinal membranes causes tractional retinal detachment (TRD) in proliferative vitreoretinopathy (PVR). The Rho-A/Rho-associated kinase signaling pathway is a principal mediator of contractile force generation in nonmuscle cells. In the current study, the relation between the Rho-kinase pathway and alpha-SMA expression and type I collagen gel contractile activity in retinal pigment epithelial (RPE) cells was investigated, using Y27632, a specific inhibitor of p160ROCK, and the involvement of the Rho-kinase pathway was evaluated in a rabbit PVR model with cultured RPE cells and platelet-rich plasma (PRP). METHODS: RPE cells were obtained from rabbits and cultured. The number of passages and the effect of Y27632 on alpha-SMA expression were studied by immunohistochemistry and immunoblot analysis. An in vitro type I collagen gel contraction assay and MTT assay evaluated the effect of Y27632 on RPE cell contractile force and proliferation. Cultured sixth-passage rabbit RPE cells were coinjected with PRP intravitreally, followed by 50 micro M of Y27632, injected weekly. The presence of TRD was assessed until 28 days to evaluate the effect of Y27632 in vivo. RESULTS: Expression of alpha-SMA was increased according to the passages. Y27632 suppressed alpha-SMA expression in cultured RPE cells and impaired contractile force. Y27632 did not affect the proliferative potential. Y27632 significantly (P < 0.01) reduced TRD development. CONCLUSIONS: Y27632 decreased alpha-SMA expression and the contractile force generated by RPE cells and attenuated PVR, indicating the involvement of the Rho-kinase pathway in cell-dependent collagen contraction in vitro and in vivo. The drug may affect the biological event by inhibiting alpha-SMA expression, and Y27632 could be useful for preventing PVR.