NF-kappaB-dependent induction of osteoprotegerin by Porphyromonas gingivalis in endothelial cells

Porphyromonas gingivalis is a major etiological pathogen of adult periodontitis characterized by alveolar bone resorption. Vascular endothelial cells supply many inflammatory cytokines into periodontal tissue. However, whether the cells contribute to bone metabolism in periodontitis remains unknown. In this study, we investigated the effect of P. gingivalis on osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) production, both of which are key regulators of bone metabolism, in human microvascular endothelial cells (HMVECs). We showed that P. gingivalis upregulated expression of OPG but not RANKL mRNA in HMVEC. P. gingivalis induced NF-kappaB activation, and the induction of OPG in HMVEC by the pathogen was blocked by the inhibitors of NF-kappaB. In addition, incubation of OPG with P. gingivalis supernatant resulted in loss of the protein. These results indicate that P. gingivalis-stimulated HMVEC secrete OPG via a NF-kappaB-dependent pathway, while the OPG is partly degraded by the bacteria. Thus, microvascular endothelial cells can act as a source of OPG and thereby may play an important role in regulating bone metabolism in periodontitis.     

Porphyromonas gingivalis infection and cell death in human aortic endothelial cells

Porphyromonas gingivalis is a periodontal pathogen that promotes a proatherogenic response in endothelial cells. Cell death responses of human aortic endothelial cells to P. gingivalis at various multiplicities of infection (MOI) were investigated by assessment of cell detachment, histone-associated DNA fragmentation, lactate dehydrogenase release and ADP:ATP ratio. Porphyromonas gingivalis at MOI 1:10-1:100 did not have a cytotoxic effect, but induced apoptotic cell death at MOI 1:500 and 1:1000. Monocyte chemoattractant protein-1 production was significantly enhanced by P. gingivalis at MOI 1:100. At higher MOI, at least in vitro, P. gingivalis mediates endothelial apoptosis, thereby potentially amplifying proatherogenic mechanisms in the perturbed vasculature.     

Arginine-specific gingipain A from Porphyromonas gingivalis induces Weibel-Palade body exocytosis and enhanced activation of vascular endothelial cells through protease-activated receptors

Gingipains, cysteine proteases derived from Porphyromonas gingivalis, are important virulence factors in periodontal diseases. We found that arginine-specific gingipain A (RgpA) increased the responsiveness of vascular endothelial cells to P. gingivalis lipopolysaccharides (LPS) and P. gingivalis whole cells to induce enhanced IL-8 production through protease-activated receptors (PARs) and phospholipase C (PLC) gamma. We therefore investigated whether RgpA-induced enhanced cell activation is mediated through exocytosis of Weibel-Palade bodies (WPBs) because they store vasoactive substances. RgpA rapidly activated PAR- and PLCgamma-dependent WPB exocytosis. In addition, angiopoietin (Ang)-2, a substance of WPB, enhanced IL-8 production by P. gingivalis LPS, suggesting that Ang-2 mediates the RgpA-induced enhanced cell responses. Thus, we propose a novel role for RgpA in induction of a proinflammatory event through PAR-mediated WPB exocytosis, which may be an important step for enhanced endothelial responses to P. gingivalis.     

Porphyromonas gingivalis-mediated shedding of extracellular matrix metalloproteinase inducer (EMMPRIN) by oral epithelial cells: a potential role in inflammatory periodontal disease

Extracellular matrix metalloproteinase inducer (EMMPRIN) or CD147 is a transmembrane glycoprotein expressed by various cell types, including oral epithelial cells. Recent studies have brought evidence that EMMPRIN plays a role in periodontitis. In the present study, we investigated the effect of Porphyromonas gingivalis, a major pathogen in chronic periodontitis, on the shedding of membrane-anchored EMMPRIN and on the expression of the EMMPRIN gene by oral epithelial cells. A potential contribution of shed EMMPRIN to the inflammatory process of periodontitis was analyzed by evaluating the effect of recombinant EMMPRIN on cytokine and matrix metalloproteinase (MMP) secretion by human gingival fibroblasts. ELISA and immunofluorescence analyses revealed that P. gingivalis mediated the shedding of epithelial cell-surface EMMPRIN in a dose- and time-dependent manner. Cysteine proteinase (gingipain)-deficient P. gingivalis mutants were used to demonstrate that both Arg- and Lys-gingipain activities are involved in EMMPRIN shedding. Real-time PCR showed that P. gingivalis had no significant effect on the expression of the EMMPRIN gene in epithelial cells. Recombinant EMMPRIN induced the secretion of IL-6 and MMP-3 by gingival fibroblasts, a phenomenon that appears to involve mitogen activated protein kinases. The present study brought to light a new mechanism by which P. gingivalis can promote the inflammatory response during periodontitis.     

Matrix attachment regulates Fas-induced apoptosis in endothelial cells: a role for c-flip and implications for anoikis

Survival of endothelial cells is critical for cellular processes such as angiogenesis. Cell attachment to extracellular matrix inhibits apoptosis in endothelial cells both in vitro and in vivo, but the molecular mechanisms underlying matrix-induced survival signals or detachment-induced apoptotic signals are unknown. We demonstrate here that matrix attachment is an efficient regulator of Fas-mediated apoptosis in endothelial cells. Thus, matrix attachment protects cells from Fas-induced apoptosis, whereas matrix detachment results in susceptibility to Fas-mediated cell death. Matrix attachment modulates Fas-mediated apoptosis at two different levels: by regulating the expression level of Fas, and by regulating the expression level of c-Flip, an endogenous antagonist of caspase-8. The extracellular signal-regulated kinase (Erk) cascade functions as a survival pathway in adherent cells by regulating c-Flip expression. We further show that detachment-induced cell death, or anoikis, itself results from activation of the Fas pathway by its ligand, Fas-L. Fas-L/Fas interaction, Fas-FADD complex formation, and caspase-8 activation precede the bulk of anoikis in endothelial cells, and inhibition of any of these events blocks anoikis. These studies identify matrix attachment as a survival factor against death receptor-mediated apoptosis and provide a molecular mechanism for anoikis and previously observed Fas resistance in endothelial cells.     

Friendly fire against neutrophils: proteolytic enzymes confuse the recognition of apoptotic cells by macrophages

Physiologically the only acceptable fate for almost all damaged or unwanted cells is their apoptotic death, followed by engulfment of the corpses by healthy neighbors or professional phagocytes. Efficient clearance of cells that have succumbed to apoptosis is crucial for normal tissue homeostasis, and for the modulation of immune responses. The disposal of apoptotic cells is finely regulated by a highly redundant system of receptors, bridging molecules and 'eat me' signals. The complexity of the system is reflected by the term: 'engulfment synapse', used to describe the interaction between a phagocytic cell and its target. In healthy humans, dying neutrophils are the most abundant and important targets for such recognition and engulfment. In inflammation the scope and importance of this complicated task is further increased. Paradoxically, despite growing evidence highlighting the priority of neutrophils clearance, the recognition of these cells by phagocytes is not as well understood as the recognition of other apoptotic cell types. New findings indicate that the interaction of phosphatidylserine (PS) on apoptotic neutrophils with its receptor on macrophages is not as critical for the specific clearance of neutrophil corpses it was previously believed. In this review we focus on recent findings regarding alternative, PS-independent "eat me" signals expressed on neutrophils during cell death and activation. Based on our own research, we emphasize the clearance of dying neutrophils, especially at the focus of bacterial infection; and the associated inflammatory reaction, which occurs in a highly proteolytic milieu containing both host and bacteria-derived proteinases. In these environments, eat-me signals expressed by neutrophils are drastically modified; arguing against the phospholipid-based detection of apoptotic cells, but supporting the importance of proteinaceous ligand(s) for the recognition of neutrophils by macrophages. In this context we discuss the effect of the gingipain R (Rgp) proteinases from Porphyromonas gingivalis on neutrophils interactions with macrophages. Since the recognition of apoptotic neutrophils is an important fundamental process, serving multiple functions in the regulation of immunity and homeostasis, we hypothesize that many pathogenic bacteria may have developed similar strategies to confuse macrophage-neutrophil interaction as a common pathogenic strategy.     

Autophagy: a highway for Porphyromonas gingivalis in endothelial cells

P. gingivalis, an important periodontal pathogen associated with adult periodontitis and a likely contributing factor to atherosclerosis and cardiovascular disease, traffics in endothelial cells via the autophagic pathway. Initially, P. gingivalis rapidly adheres to the host cell surface followed by internalization via lipid rafts and incorporation of the bacterium into early phagosomes. P. gingivalis activates cellular autophagy to provide a replicative niche while suppressing apoptosis. The replicating vacuole contains host proteins delivered by autophagy that are used by this asaccharolytic pathogen to survive and replicate within the host cell. When autophagy is suppressed by 3-methyladenine or wortmannin, internalized P. gingivalis transits to the phagolysosome where it is destroyed and degraded. Therefore, the survival of P. gingivalis depends upon the activation of autophagy and survival of the endothelial host cell, but the mechanism by which P. gingivalis accomplishes this remains unclear.     

Autophagy: A Highway for Porphyromonas gingivalis in Endothelial Cells

P. gingivalis, an important periodontal pathogen associated with adult periodontitis and a likely contributing factor to atherosclerosis and cardiovascular disease, traffics in endothelial cells via the autophagic pathway. Initially, P. gingivalis rapidly adheres to the host cell surface followed by internalization via lipid rafts and incorporation of the bacterium into early phagosomes. P. gingivalis activates cellular autophagy to provide a replicative niche while suppressing apoptosis. The replicating vacuole contains host proteins delivered by autophagy that are used by this asaccharolytic pathogen to survive and replicate within the host cell. When autophagy is suppressed by 3-methyladenine or wortmannin, internalized P. gingivalis transits to the phagolysosome where it is destroyed and degraded. Therefore, the survival of P. gingivalis depends upon the activation of autophagy and survival of the endothelial host cell, but the mechanism by which P. gingivalis accomplishes this remains unclear.     

The role of osteoprotegerin and tumor necrosis factor-related apoptosis-inducing ligand in human microvascular endothelial cell survival

Endothelial cell survival and antiapoptotic pathways, including those stimulated by extracellular matrix, are critical regulators of vasculogenesis, angiogenesis, endothelial repair, and shear-stress-induced endothelial activation. One of these pathways is mediated by alpha(v)beta(3) integrin ligation, downstream activation of nuclear factor-kappaB, and subsequent up-regulation of osteoprotegerin (OPG). In this study, the mechanism by which OPG protects endothelial cells from death was examined. Serum-starved human microvascular endothelial cells (HMECs) plated on the alpha(v)beta(3) ligand osteopontin were protected from cell death. Immunoprecipitation experiments indicated that OPG formed a complex with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in HMECs under these conditions. Furthermore, inhibitors of TRAIL, including recombinant soluble TRAIL receptors and a neutralizing antibody against TRAIL, blocked apoptosis of serum-starved HMECs plated on the nonintegrin attachment factor poly-d-lysine. Whereas TRAIL was unable to induce apoptosis in HMECs plated on osteopontin, the addition of recombinant TRAIL did increase the percentage of apoptotic HMECs plated on poly-d-lysine. This evidence indicates that OPG blocks endothelial cell apoptosis through binding TRAIL and preventing its interaction with death-inducing TRAIL-receptors     

Protein kinase B inhibits endostatin-induced apoptosis in HUVECs

Endostatin is a tumor-derived angiogenesis inhibitor, and the endogenous 20 kDa carboxyl-terminal fragment of collagen XVIII. In addition to inhibiting angiogenesis,endostatin inhibits tumor growth and the induction of apoptosis in several endothelial cell types. However, the mechanisms that regulate endostatin-induced apoptotic cell death are unclear. Here, we investigated apoptotic cell death and the underlying regulatory mechanisms elicited of endostatin in human umbilical vein endothelial cells (HUVECs). Endostatin was found to induce typical apoptotic features, such as, chromatin condensation and DNA fragmentation in these cells. Thus, as the phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in various cell types, we investigated whether this pathway could protect cells against endostatin induced apoptosis. It was found that the inhibition of PI3K/PKB significantly increased endostatin-induced apoptosis, and that endostatininduced cell death is physiologically linked to PKB-mediated cell survival through caspase-8.     

Apoptotic neutrophils undergoing secondary necrosis induce human lung epithelial cell detachment

Clearance of apoptotic neutrophils by alveolar macrophages plays an important role in the resolution phase of lung inflammation. If not cleared, apoptotic neutrophils are postulated to release histotoxic granular contents. Since numerous cellular proteins are degraded during apoptosis, we sought to determine whether functional serine proteinases are indeed released by apoptosing neutrophils in vitro. In a coculture system, cytokine-activated neutrophils induced detachment in the human epithelial cell line, A549. This process was CD18- and serine proteinase-dependent. Early apoptotic neutrophils induced significant detachment, but live, senescent, resting neutrophils and terminal, secondary necrotic neutrophils had a different effect. This detachment process was CD18-independent but serine proteinase-dependent. Similarly, detachment occurred with primary human small airway epithelial cells. Notably, epithelial cell detachment correlated with the transition of early apoptotic neutrophils to secondary necrosis and with the accumulation of elastase in the supernatant. The membrane integrity of lung epithelial cells was damaged in advance of significant cell detachment. These observations suggest that not only live activated neutrophils but also apoptosing neutrophils can reveal functional elastase activities. Furthermore, the rapidity of the transition emphasizes the importance of the prompt clearance of apoptotic neutrophils before they progress to secondary necrosis at the site of lung inflammation.C.Y.L. and Y.H.L contributed equally to the work on this project as first authors.     

Porphyromonas gingivalis influences actin degradation within epithelial cells during invasion and apoptosis

Porphyromonas gingivalis, a Gram-negative oral pathogen, has been shown to induce apoptosis in human gingival epithelial cells, yet the underlining cellular mechanisms controlling this process are poorly understood. We have previously shown that the P. gingivalis proteases arginine and lysine gingipains, are necessary and sufficient to induce host cell apoptosis. In the present study, we demonstrate that 'P. gingivalis-induced apoptosis' is mediated through degradation of actin leading to cytoskeleton collapse. Stimulation of human gingival epithelial cells with P. gingivalis strains 33277 and W50 at moi:100 induced β-actin cleavage as early as 1 h and human serum inhibited this effect. By using gingipain-deficient mutants of P. gingivalis and purified gingipains, we demonstrate that lysine gingipain is involved in actin hydrolysis in a dose and time-dependent manner. Use of Jasplakinolide and cytochalasin D revealed that P. gingivalis internalization is necessary for actin cleavage. Further, we also show that lysine gingipain from P. gingivalis can cleave active caspase 3. Taken together, we have identified actin as a substrate for lysine gingipain and demonstrated a novel mechanism involved in microbial host cell invasion and apoptosis.     

P38 mitogen-activated protein kinase pathways are involved in the hypertrophy and apoptosis of cardiomyocytes induced by Porphyromonas gingivalis conditioned medium

The surrounding medium of periodontal pathogen Porphyromonas gingivalis (P. gingivalis) increased cardiomyocyte hypertrophy and apoptosis whereas Actinobaeillus actinomycetemcomitans and Prevotella intermedia had no effects. The purpose of this study is to clarify the role of p38 pathway in P. gingivalis conditioned medium-induced H9c2 myocardial cell hypertrophy and apoptosis. DNA fragmentation, cellular morphology, nuclear condensation, p38 protein products, and mitochondrial-dependent apoptotic related proteins in cultured H9c2 myocardial cell were measured by agarose gel electrophoresis, immunofluorescence, DAPI, and western blotting following P. gingivalis conditioned medium and/or pre-administration of SB203580 (p38 inhibitor). The p38 protein products and associated activities in H9c2 cells were both upregulated by P. gingivalis conditioned medium. P. gingivalis conditioned medium increased cellular sizes, DNA fragmentation, nuclear condensation, mitochondrial Bcl2-associated death promoter (Bad), cytosolic cytochrome c (cyt c), and the activated form of caspase-9 proteins in H9c2 cells. The increased cellular sizes, DNA fragmentation, nuclear condensation, Bad, cyt c, and caspase-9 activities of H9c2 cells treated with P. gingivalis conditioned medium were all significantly reduced after pre-administration of SB203580. Our findings suggest that the activity of p38 signal pathway may be initiated by P. gingivalis conditioned medium and further activate mitochondrial-dependent apoptotic pathways leading to cell death in cultured H9c2 myocardial cells.     

Arginine-specific gingipains from Porphyromonas gingivalis stimulate production of hepatocyte growth factor (scatter factor) through protease-activated receptors in human gingival fibroblasts in culture

Cystein proteinases (gingipains) from Porphyromonas gingivalis cleave a broad range of in-host proteins and are considered to be key virulence factors in the onset and development of adult periodontitis and host defense evasion. In periodontitis, an inflammatory disease triggered by bacterial infection, the production of hepatocyte growth factor (HGF) is induced not only by various factors derived from the host, such as inflammatory cytokines, but also by bacterial components. In this study we examined the possible enhanced production of HGF produced by human gingival fibroblasts upon stimulation with gingipains. Arginine-specific gingipain (Rgp) caused a marked production of HGF into the supernatant, the induction of HGF expression on the cell surface, and the up-regulation of HGF mRNA expression in a dose-dependent and an enzymatic activity-dependent manner. Because it has been reported that Rgp activated protease-activated receptors (PARs), we examined whether the induction of HGF triggered by Rgps on human gingival fibroblasts occurred through PARs. An RNA interference assay targeted to PAR-1 and PAR-2 mRNA revealed that gingipains-induced secretion of HGF was significantly inhibited by RNA interference targeted to PAR-1 and PAR-2. In addition, the Rgps-mediated HGF induction was completely inhibited by the inhibition of phospholipase C and was clearly inhibited by RNA interference targeted to p65, which is an NF-kappaB component. These results suggest that Rgps activated human gingival fibroblasts to secrete HGF in the inflamed sites and the mechanism(s) involved may actively participate in both inflammatory and reparative processes in periodontal diseases.     

Activation of the endothelial nitric-oxide synthase by tumor necrosis factor-alpha. A novel feedback mechanism regulating cell death

Cell death via apoptosis induced by tumor necrosis factor-alpha (TNF-alpha) plays an important role in many physiological and pathological conditions. The signal transduction pathway activated by this cytokine is known to be regulated by several intracellular messengers. In particular, in many systems nitric oxide (NO) has been shown to protect cells from TNF-alpha-induced apoptosis. However, whether NO can be generated by the cytokine to down-regulate its own apoptotic program has never been studied. We have addressed this question in HeLa Tet-off cell clones stably transfected with the endothelial NO synthase under a tetracycline-responsive promoter. Endothelial NO synthase, induced about 100-fold in these cells by removal of the antibiotic, retained the characteristics of the native enzyme of endothelial cells, both in terms of intracellular localization and functional activity. Expression of the endothelial NO synthase was sufficient to protect from TNF-alpha-induced apoptosis. This protection was mediated by the generation of NO. TNF-alpha itself stimulated endothelial NO synthase activity to generate NO through a pathway involving its lipid messenger, ceramide. Our results identify a novel mechanism of regulation of a signal transduction pathway activated by death receptors and suggest that NO may constitute a built-in mechanism by which TNF-alpha controls its own apoptotic program.     

Gingipains inactivate a cell surface ligand on Porphyromonas gingivalis that induces TLR2-and TLR4-independent signaling

Arginine-specific gingipain and lysine-specific gingipain are two major cysteine proteinases produced by Porphyromonas gingivalis. To clarify the role of gingipains in the interaction between P. gingivalis and the innate immune system, CHO reporter cells expressing TLR2 or TLR4 were stimulated with wildtype or gingipain-deficient P. gingivalis cells and activation of nuclear factor-kappaB in these cells was examined. While CHO/CD14 cells and 7.19 cells, an MD-2-defective mutant derived from CHO/CD14 cells, failed to respond to wild-type P. gingivalis, they responded to gingipain-deficient P. gingivalis. On the other hand, CHO/CD14/TLR2 cells responded to both wild-type and gingipain-deficient P. gingivalis. These results suggested that gingipains have no effects on TLR2-dependent signaling from P. gingivalis but have inhibitory effects on TLR2-and TLR4-independent signaling in CHO cells. Indeed, the activity of gingipain-deficient P. gingivalis to induce the activation of 7.19 cells was diminished after treatment of the bacterial cells with gingipains. We next partially purified bacterial cell components activating 7.19 cells from gingipain-deficient P. gingivalis. The activity of the partially purified components was diminished by treatment with heat or gingipains. It is also noteworthy that anti-CD14 mAb inhibited the activation of 7.19 cells induced by the partially purified components. These results indicated that the components of P. gingivalis that were able to induce TLR2-and TLR4-independent signaling were inactivated by gingipains before being recognized by CD14. The inactivation of the components would be helpful for P. gingivalis to escape from the innate immune system.     

Role of protease-activated receptor-2 in inflammation, and its possible implications as a putative mediator of periodontitis

Proteinase-activated receptor-2 (PAR2) belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. This receptor is widely distributed throughout the body and seems to be importantly involved in inflammatory processes. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and bacterial proteases, such as gingipain produced by Porphyromonas gingivalis. This review describes the current stage of knowledge of the possible mechanisms that link PAR2 activation with periodontal disease, and proposes future therapeutic strategies to modulate the host response in the treatment of periodontitis.     

VEGF prevents apoptosis of human microvascular endothelial cells via opposing effects on MAPK/ERK and SAPK/JNK signaling

Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, promotes endothelial cell survival and angiogenesis. We recently showed that VEGF can support the growth of human dermal microvascular endothelial cells (HDMEC) and human umbilical vein endothelial cells in serum-free medium. Reasoning that VEGF might be modulating apoptotic signal transduction pathways, we examined mechanisms involved in the anti-apoptotic effect of VEGF on starvation- and ceramide-induced apoptosis in HDMEC. We observed that VEGF ameliorated the time-dependent increase in apoptosis, as demonstrated by morphologic observations, TUNEL assay, and DNA fragmentation. On the other hand, basic fibroblast growth factor only partially prevented apoptosis in serum-starved HDMEC; platelet-derived growth factor-BB was completely ineffective. VEGF activated the phosphorylation of extracellular signal regulated kinase (ERK)1 (p44 mitogen-activated protein kinase; MAPK) and ERK2 (p42 MAPK) in a time- and concentration-dependent manner. Both the VEGF-induced activation and its anti-apoptotic effect were prevented by the specific MAPK/ERK inhibitor PD98059. The presence of VEGF also inhibited the sustained activation of stress-activated protein kinase/c-jun-NH2-kinase (SAPK/JNK) caused by serum starvation and ceramide treatment. Activation of the MAPK pathway together with inhibition of SAPK/JNK activity by VEGF appears to be a key event in determining whether an endothelial cell survives or undergoes programmed cell death.     

Bone marrow mesenchymal stem cells enhance autophagy and help protect cells under hypoxic and retinal detachment conditions

Our study aimed to evaluate the protective role and mechanisms of bone marrow mesenchymal stem cells (BMSCs) in hypoxic photoreceptors and experimental retinal detachment. The cellular morphology, viability, apoptosis and autophagy of hypoxic 661w cells and cells cocultured with BMSCs were analysed. In retinal detachment model, BMSCs were intraocularly transplanted, and then, the retinal morphology, outer nuclear layer (ONL) thickness and rhodopsin expression were studied as well as apoptosis and autophagy of the retinal cells. The hypoxia-induced apoptosis of 661w cells obviously increased together with autophagy levels increasing and peaking at 8 hours after hypoxia. Upon coculturing with BMSCs, hypoxic 661w cells had a better morphology and fewer apoptosis. After autophagy was inhibited, the apoptotic 661w cells under the hypoxia increased, and the cell viability was reduced, even in the presence of transplanted BMSCs. In retina-detached eyes transplanted with BMSCs, the retinal ONL thickness was closer to that of the normal retina. After transplantation, apoptosis decreased significantly and retinal autophagy was activated in the BMSC-treated retinas. Increased autophagy in the early stage could facilitate the survival of 661w cells under hypoxic stress. Coculturing with BMSCs protects 661w cells from hypoxic damage, possibly due to autophagy activation. In retinal detachment models, BMSC transplantation can significantly reduce photoreceptor cell death and preserve retinal structure. The capacity of BMSCs to reduce retinal cell apoptosis and to initiate autophagy shortly after transplantation may facilitate the survival of retinal cells in the low-oxygen and nutrition-restricted milieu after retinal detachment.