Progression Of Proliferative Vitreoretinopathy Research Articles

Использование аутологичной кондиционированной плазмы (аср) в хирургии тяжелой патологии заднего отрезка глаза. Трехлетний опыт использования технологии

Purpose. To evaluate the effectiveness and long-term results of the use of autologous conditioned plasma in the surgery of open eye injury, including perforating eye injuries, surgery of the cicatricial stage of the subretinal neovascular membrane, rhematogenous retinal detachment, including retinal detachment with macular hole in people with high myopia with expressed scleral staphyloma, without the use of an additional laser coagulation of the retina and the almost complete exclusion of silicone tamponade. Material and methods. Since 2018, more than 250 patients with various vitreous pathologies of the posterior eye segment have been operated on perforating eye injuries, cicatricial stage of the subretinal neovascular membrane, idiopathic macular rupture, rhegmatogenous retinal detachment, including those with macular holes, including cases with high myopia and expressed scleral staphyloma. The developed technology of surgical closure of defects of the retina, choroid, sclera of various lengths using autologous conditioned plasma, made it possible to completely eliminate the need for laser coagulation of the retina, which in most cases would be difficult and unsafe, and to practically exclude silicone tamponade. A method of seamless sealing of sclero and conjunctivotomies with the variant of air tamponade using autologous conditioned plasma has been developed and successfully applied. Results. In the early postoperative period the patients in all clinical groups had no complications. Complete adhesion of the retina and sealing of defects in the structures of the eye after using autologous conditioned plasma without additional laser coagulation of the retina on the background of the applied air and silicone tamponade was observed in all patients. In the long-term follow-up period up to 28 months, there were isolated recurrences of retinal detachment after surgery of complex open eye injury, rhematogenous retinal detachment on the background of progression of proliferative vitreoretinopathy. More than 89% of patients had no complications in the long-term period. Complete closure of the sclero and conjunctivotomies without the use of additional suture fixation had been achieved. In all cases, the vascular pattern of the conjunctiva was preserved, in no case was a reactive process detected at the site of application of autologous conditioned plasma. Conclusion. The use of autologous conditioned plasma is an effective and safe method of sealing defects of the retina, choroid, sclera of various lengths in complex open eye injury, of surgery of rhematogenous retinal detachment, including high myopia and the presence of a macular hole, of surgery of the cicatricial stage of the subretinal neovascular membrane, allowing to completely eliminate the need for laser coagulation of the retina, minimize the need for silicone tamponade.

The Proteome of Preretinal Tissue in Proliferative Vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) is the leading cause of retinal detachment repair failure. However, the molecular pathogenesis remains incompletely understood. Determining the proteome of PVR will help to identify novel therapeutic targets. Preretinal tissue samples, delaminated during surgery from six PVR cases and one idiopathic epiretinal membrane (ERM) were analyzed by mass spectrometry. Tandem mass spectra were extracted using the UniProt database, generating a list of 896 proteins, which were subjected to pathway set and fold-change (ERM vs PVR) analyses. Two pathways were enriched in PVR: extracellular matrix (ECM) organization and extracellular structure organization. A fold-change analysis comparing mean total spectral counts from PVR to an ERM control identified fibronectin, the ECM glycoprotein, as the protein most significantly elevated in PVR compared to ERM. These data identify pathwayskey to PVR progression, including thoseinvolved in cell-mediated ECM assembly and thus tractional force generation at the cellular level. [Ophthalmic Surg Lasers Imaging Retina. 2021;52:S5-S12.].

Targeting matrix stiffness-induced activation of retinal pigment epithelial cells through the RhoA/YAP pathway ameliorates proliferative vitreoretinopathy

The purpose of this study was to investigate whether excessive extracellular matrix (ECM) deposition-induced mechanical matrix stiffness plays a key role in promoting retinal pigment epithelial (RPE) cell activation and the subsequent development of proliferative vitreoretinopathy (PVR). Human ARPE-19 cells were cultured on either 50 kappa (stiff) or 0.5 kappa (soft) gel-coated coverslips. Reverse and knockdown experiments were carried out to establish a model of matrix stiffness-induced activation in ARPE-19 cells in vitro. A PVR mouse model was established by the intravitreal injection of dispase. The effects of RhoA/YAP signalling blockade on matrix stiffness-induced ARPE-19 cell activation and PVR-induced retinal fibrosis were determined by using a combination of the Yes-associated protein (YAP) inhibitor verteporfin and the RhoA inhibitor C3 exoenzyme. Matrix stiffness stimulated YAP nuclear translocation and expression in ARPE-19 cells. The effect of YAP activation was dependent on F-actin cytoskeleton polymerization and RhoA activity, forming the RhoA/YAP signalling pathway. Upstream pharmacological blockade of RhoA by C3 exoenzyme or downstream blockade of YAP by verteporfin reduced the invasion, migration, and MMP expression of ARPE-19 cells and collagen gel contraction. Furthermore, blockade of RhoA/YAP signalling reduced PVR-induced retinal fibrogenesis and inhibited the TGF-β/Smad pathway in vivo. RhoA/YAP signalling modulates matrix stiffness-induced activation of ARPE-19 cells. Targeting this signalling pathway could alleviate PVR-induced retinal fibrosis and suggests attractive novel therapeutic strategies for intervening in the progression of PVR.

Экстрасклеральное пломбирование с использованием эндоиллюминации: первый опыт

Purpose. To study the effectiveness of extrascleral filling (ESF) using endoillumination in the surgical treatment of patients with primary rhegmatogenous retinal detachment (RRD). Material and methods. The material for the study was the data of a comprehensive clinical examination and surgical treatment by ESF method using endoillumination in 17 patients (17 eyes) with RRD. In 7 cases (41%), the macular area was involved in the detachment process. In 5 cases (29.4%), local scleral filling was performed. In the remaining 12 cases (70.6%), the local ESF was supplemented with a circling silicone element. Surgical intervention was supplemented by transscleral drainage of subretinal fluid (SRF) in 10 cases (59%) and pneumatic retinopexy with SF6 gas 50% in 8 cases (48%). Results. In the general group of patients, best corrected visual acuity (BCVA) increased from 0.35 to 0.46. In the subgroup of patients with a detached macular area, the positive dynamics is more pronounced, BCVA increased from 0.1 to 0.28. The progression of proliferative vitreoretinopathy caused the recurrence of retinal detachment in two patients (11.8%). Recurrences were diagnosed after 3 and 5 months, respectively. In both cases, a vitrectomy with tamponade of the vitreous cavity with silicone oil 5000 Cst was performed. The percentage of successful anatomical outcome after the first operation in our study was 82%. The percentage of successful achievement of the final anatomical result was 94%. In two cases, additional injection of SF6 gas into the vitreous cavity was required. Conclusion. This type of surgical treatment is an effective method of surgical treatment of patients with RRD. In our study, the successful anatomical outcome after the first operation was recorded in 82% of patients, which correlates with the data of the authors who also used this method (83–92%). Surgeons who performed surgical treatment using this technique in our study note improved workplace ergonomics when visualizing the fundus using an operating microscope and endoillumination compared with indirect ophthalmoscopy. Other teams of authors came to this conclusion as well. In our study, there were no complications associated with the introduction of a light pipe into the vitreous cavity (iatrogenic crystalline lens injury, endophthalmitis), which indicates the safety of this type of surgical treatment.

A Mouse Model of Proliferative Vitreoretinopathy Induced by Intravitreal Injection of Gas and RPE Cells.

PurposeDevelop a reproducible proliferative vitreoretinopathy (PVR) mouse model that mimics human PVR pathology.MethodsMice received intravitreal injections of SF6 gas, followed by retinal pigment epithelial cells 1 week later. PVR progression was monitored using fundus photography and optical coherence tomography imaging, and histologic analysis of the retina as an endpoint. We developed a PVR grading scheme tailored for this model.ResultsWe report that mice that received gas before retinal pigment epithelial injection developed more severe PVR. In the 1 × 104 retinal pigment epithelial cell group, after 1 week, 0 of 11 mice in the no gas group developed grade 4 or greater PVR compared with 5 of 13 mice in the SF6 gas group (P = 0.02); after 4 weeks, 3 of 11 mice in the no gas group developed grade 5 or greater PVR compared with 11 of 14 mice in the SF6 gas group (P = 0.01). We were able to visualize contractile membranes both on the retinal surface as well as within the vitreous of PVR eyes, and demonstrated through immunohistochemical staining that these membranes expressed fibrotic markers alpha smooth muscle actin, vimentin, and fibronectin, as well as other markers known to be found in human PVR membranes.ConclusionsThis mouse PVR model is reproducible and mimics aspects of PVR pathology reported in the rabbit PVR model and human PVR. The major advantage is the ability to study PVR development in different genetic backgrounds to further elucidate aspects of PVR pathogenesis. Additionally, large-scale experiments for testing pharmacologic agents to treat and prevent PVR progression is more feasible compared with other animal models.Translational RelevanceThis model will provide a platform for screening potential drug therapies to treat and prevent PVR, as well as elucidate different molecular pathways involved in PVR pathogenesis.

Improving outcomes in retinal detachment: the potential role of rho-kinase inhibitors.

Retinal detachment initiates a series of events that lead to degenerative changes in retinal synaptic architecture as well as the well-known phenomena of gliosis and photoreceptor apoptosis. Retinal reattachment does not always result in complete visual recovery, even if the fovea is not directly involved in the detachment. Rho-kinase (ROCK) inhibitors may mitigate some of these deleterious changes including disruption of synaptic architecture, photoreceptor apoptosis, and initiation of the epithelial-mesenchymal transition that characterizes proliferative vitreoretinopathy (PVR). This review focuses on the use of ROCK inhibitors to modulate synaptic disjunction. ROCK inhibition prevents retinal detachment-induced photoreceptor synaptic terminal retraction (i.e., synaptic disjunction), thereby diminishing the damage of the first synapse in the visual pathway. ROCK inhibition also reduces retinal detachment-induced photoreceptor apoptosis and suppresses PVR progression in preclinical models. Inhibition of ROCK may help to optimize visual recovery after retinal detachment surgery or iatrogenic detachments during cell transplantation or viral subretinal injection and might play a role in reducing the risk of PVR after retinal detachment surgery.

SURGICAL OUTCOMES OF 25-GAUGE PARS PLANA VITRECTOMY USING AIR AS AN INTERNAL TAMPONADE FOR PRIMARY RHEGMATOGENOUS RETINAL DETACHMENT.

To report surgical outcomes of 25-gauge pars plana vitrectomy using air as an internal tamponade for patients with primary rhegmatogenous retinal detachment (RRD). A retrospective clinical study of 59 eyes of 59 consecutive patients presented with primary RRD at the Beijing Tongren Eye Center in China. From August 2016 to May 2018, medical records of the patients who underwent 25-gauge pars plana vitrectomy with air tamponade for RRD were reviewed. The main outcome measures were primary and final anatomical success (retinal re-attachment) rates, and postoperative complications. Of the 59 patients, aged 54.47 ± 11.81 years, 31 (52.5%) were men. Vitrectomy was performed 3 to 40 (averaged 16.98 ± 10.17) days after the onset of symptoms, and the mean follow-up period was 12.90 ± 5.92 months (ranging 6.07-26.10 months). Forty-two eyes (71.2%) had RRD with retinal breaks in the superior half of the retina, and the mean number of retinal breaks was 1.75 ± 0.94. Three eyes (5.1%) had RRD with giant retinal tears. Of the 59 eyes, 35 (59.3%) had RRD with inferior quadrants involved. Proliferative vitreoretinopathy (PVR) gradings were C1 in 2 (3.4%) eyes and B or below in 57 (96.6%) eyes. The primary and final anatomical success rates were 94.9% (56/59) and 98.3% (58/59), respectively. Of the three eyes which developed re-detachment of the retina, one eye had postoperative progression of PVR and two eyes were RRD associated with macular hole in high myopia. Postoperative complications included 5 eyes (8.5%) with serous choroidal detachment within 3 days after surgery and 4 eyes (6.8%) with macular epiretinal membrane formation 1 to 8 months after surgery. Secondary cataract surgery was performed in 13 of the 53 phakic eyes (24.5%) during follow-up. Small-gauge pars plana vitrectomy with air tamponade may be effective in treating selected cases of relatively simple primary RRD. Additional studies are needed to verify the efficacy of this surgical approach for more complicated cases such as those with giant retinal tears.

Vitreous levels of Lipocalin-2 on patients with primary rhegmatogenous retinal detachment.

PurposeTo measure vitreous levels of Lipocalin2 (LCN2) in patients with rhegmatogenous retinal detachment (RRD) and investigate potential association with presence of proliferative vitreoretinopathy (PVR).Materials and methodsCollection of undiluted vitreous samples from 24 patients suffering from RRD and 10 control patients undergoing vitrectomy for: vitreomacular traction (VMT) (n = 2), idiopathic epiretinal membrane (iERM) (n = 6) and full thickness macular hole (FTMH) (n = 2). Quantitative analysis of LCN2 has been made with flow cytometry. Lens status, duration of symptoms, quadrants of detachment, as well as level of PVR, were assessed. Statistical analysis included Mann-Whitney test, Kruskal-Wallis test, t-test, Spearman’s correlation coefficient and Fisher's exact test.ResultsMedian LCN2 was significantly higher in the RRD group as compared to control (p<0.001). Within the RRD group there was a positive correlation between LCN2 and PVR grade (rs = 0.94, p<0.001). Median LCN2 was 35,759 pg/ml (IR = 55,347) in grade C PVR, 9,387 pg/ml (IR = 3721) in grade B, 4,917 pg/ml (IR = non computable) in grade A and 3,921 pg/ml (2132) in the no PVR group. Median LCN2 was also significantly higher in pseudophakic patients as compared to phakic patients (p = 0.007). LCN2 also correlates with the extend of detachment (≤2 vs >2 quadrants, p<0.001) as well as with duration of symptoms (rs = 0.87, p<0.001). After multivariate linear regression analysis, only PVR was independently related with LCN2 concentration. In particular, increased PVR grading was associated with increased LCN2 concentration (coefficient b = 2.97, 95% confidence interval = 1.89 to 4.67, p<0.001).ConclusionA positive correlation between vitreous levels of LCN2 and PVR grading reveals a potential role in the pathogenesis and progression of PVR. Further studies could elucidate if LCN2 could be a therapeutic target.

Biomarkers for PVR in rhegmatogenous retinal detachment.

PurposeVarious profibrotic and proinflammatory cytokines have been found upregulated in uncomplicated primary retinal detachment (pRD), but without providing a uniform picture. Here, we compare the cyto- and chemokine profiles in pRD with and without proliferative vitreoretinopathy (PVR) in an attempt to unravel relevant differences not in single cytokines, but in the cytokine profiles at diagnosis.MethodsUndiluted vitreous fluid (VF) was obtained at the beginning of surgery from 174 eyes with pRD without relevant PVR (maximally grade B; group 1; n = 81) and with moderate or advanced PVR requiring a gas tamponade (group 2; n = 49) or silicon oil filling (group 3; n = 44). VF of eyes undergoing macular hole (MH) surgery served as controls (group 4; n = 26). Forty-three cytokines were quantified in parallel using a multiplex cytokine analysis system (Bioplex). For all comparisons we applied Holm’s correction to control for multiple comparisons.Results44.9% of group 2 eyes presented grade C1 and 55.1% C2-C3, whereas 86.4% of group 3 eyes exhibited a PVR grade of C2-D.CCL19 was the only cytokine that displayed higher concentrations in the vitreous of eyes with PVR C1 compared to lower PVR grades. Eyes with PVR C2-D showed higher levels of CCL27, CXCL6, IL4, IL16, CXCL10, CCL8, CCL22, MIG/CXCL9, CCL15, CCL19, CCL 23 and CXCL12 compared to controls. Interestingly, no difference of cytokine levels was detected between C1 and C2-D PVR.ConclusionsCCL19 may represent a potential biomarker for early PVR progression that holds promise for future diagnostic and therapeutic applications.

Autophagy resists EMT process to maintain retinal pigment epithelium homeostasis.

Proliferative vitreoretinopathy (PVR) is the most serious fibrous complication that causes vision loss after intraocular surgery, and there is currently no effective treatment in clinical. Autophagy is an important cell biological mechanism in maintaining the homeostasis of tissues and cells, resisting the process of EMT. However, it is still unclear whether autophagy could resist intraocular fibrosis and prevent PVR progression. In this study, we investigated the expression of mesenchymal biomarkers in autophagy deficiency cells and found these proteins were increased. The mesenchymal protein transcription factor Twist can bind to autophagy related protein p62 and promote the degradation of Twist, which reduced the expression of mesenchymal markers. By constructing an EMT model of retinal pigment epithelial (RPE) cells in vitro, we found that autophagy was activated in the EMT process of RPE cells. Moreover, in autophagy deficient RPE cell line via knockdown autophagy related protein 7 (Atg7), the expression of epithelial marker claudin-1 was suppressed and the mesenchymal markers were increased, accompanied by an increase in cell migration and contractility. Importantly, RPE epithelial properties can be maintained by promoting autophagy and effectively reversing TFG-β2-induced RPE fibrosis. These observations reveal that autophagy may be an effective way to treat PVR.

Safety, pharmacokinetics, and prevention effect of intraocular crocetin in proliferative vitreoretinopathy

The study was designed to determine the safety and pharmacokinetics of intraocular crocetin and examine whether crocetin inhibits the development of proliferative vitreoretinopathy (PVR) in a rabbit model. In the toxicity study, the right eyes of rabbits were injected with 0.2 μmol or 0.4 μmol crocetin. The left eyes were injected with 0.1 ml phosphate buffered saline (PBS) containing the same concentration of DMSO. Fundus photography, optical coherence tomography (OCT), and electroretinogram (ERG) were obtained at baseline and 14 days. Afterward, the eyes were enucleated for histopathological analysis and terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) assay. In the pharmacokinetic study, the eyes received an intravitreous injection of 0.4 μmol crocetin to detect vitreous drug levels with HPLC at specific time points. In the efficacy study, PVR was induced with an intravitreal injection of ARPE-19 cells in rabbits. Then ten eyes were injected with 0.4 μmol crocetin, and the other 10 eyes received 0.1 ml PBS. Fundus photography, OCT and ERG were performed at days 3 and 7 and weekly for a total of 4 weeks after injection. Afterward, the eyes were enucleated and subjected to histological analysis and TUNEL staining. The results demonstrated no signs of retinal toxicity. Intravitreal injection of 0.4 μmol crocetin had a half-life of 4.231 h. Treatment with crocetin significantly inhibited the progression of PVR in parallel with a reduced expression of α-SMA, collagen fibers and Ki67. These results indicate that crocetin is an effective and safe inhibitor of PVR in rabbit models.

BMP7 antagonizes proliferative vitreoretinopathy through retinal pigment epithelial fibrosis in vivo and in vitro.

The major pathogenesis of proliferative vitreoretinopathy (PVR) is that retinal pigment epithelial (RPE) cells undergo epithelial-mesenchymal transition (EMT) because of disordered growth factors, such as TGF-β, in the vitreous humor. Bone morphogenetic proteins (BMPs) are pluripotent growth factors. In this study, we identified the antifibrotic activity of BMP7 in a PVR model both in vivo and in vitro. BMP7 expression was confirmed on the PVR proliferative membranes. BMP7 was down-regulated in the PVR vitreous humor and TGF-β-induced RPE cell EMT. In the in vivo studies, BMP7 injection attenuated PVR progression in the eyes of the rabbit model. Additionally, BMP7 treatment maintained RPE cell phenotypes and relieved TGF-β2-induced EMT, migration, and gel contraction in vitro. BMP7 inhibited the TGF-β2-induced up-regulation of fibronectin and α-smooth muscle actin and the down-regulation of E-cadherin and zona occludens-1 by balancing the TGF-β2/Smad2/3 and BMP7/Smad1/5/9 pathways. These findings provide direct evidence of the ability of BMP7 in PVR inhibition and the potential of BMP7 for use in PVR therapeutic intervention.-Yao, H., Ge, T., Zhang, Y., Li, M., Yang, S., Li, H., Wang, F. BMP7 antagonizes proliferative vitreoretinopathy through retinal pigment epithelial fibrosis in vivo and in vitro.

Knockdown of Fibromodulin Inhibits Proliferation and Migration of RPE Cell via the VEGFR2-AKT Pathway.

Purpose Recent research has provided novel insight into the function of fibromodulin (FMOD) in wound healing and angiogenesis. The role of FMOD in initiation of proliferative vitreoretinopathy (PVR) has not been studied. This study investigated the effect of FMOD on human retinal pigment epithelial (RPE) cell, which plays an essential role in the progression of PVR, and the possible mechanisms. Methods Small interfering (si) RNA-based gene transfer technology was used to decrease FMOD expression and to study its effects on RPEs in vitro. Cell Counting Kit-8 assays, transwells, and flow cytometry analysis were used to measure cell proliferation, migration, cell cycle, and apoptosis. Western blot was used to measure expression of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), extracellular signal-related kinase 1/2 (ERK1/2), and phosphoinositide 3 kinase (PI3K/AKT). Results After transfection of RPEs with a FMOD-specific siRNA, cell proliferation and migration were inhibited to the percentage of 65% ± 5% and 39% ± 10%, respectively, compared to the control group. Depletion of FMOD induced cell cycle arrest and apoptosis in RPE cells. Downregulation of VEGF, VEGFR2, and phosphorylated AKT (p-AKT) were detected in transfected RPEs. Conclusion Depletion of FMOD selectively downregulated the expression of VEGF and VEGFR2 and inhibited the signaling pathway of AKT phosphorylation, which consequently inhibited the proliferation and migration of RPE Cell.

Effect of N-acetylcysteine on the transforming growth factor β1-induced transdifferentiation of retinal pigment epithelial cell to myofibroblast

Background The transdifferentiation of retinal pigment epithelial (RPE) cell to myofibroblast is a central event in the development and progression of proliferative vitreoretinopathy (PVR). N-acetylcysteine (NAC) can inhibit the transdifferentiation of multiple cells to myofibroblasts via suppressing the production of reactive oxygen species (ROS) and the phosphorylation of MAPK induced by transforming growth factor-β1(TGF-β1), but the effect of NAC on the TGF-β1-induced transdifferentiation of human RPE cell line ARPE-19 cell to myofibroblast and its underlying molecular mechanism is still unclear. Objective This study was to investigate the effect of NAC on the TGF-β1-induced transdifferentiation of ARPE-19 cell to myofibroblast and its underlying mechanisms. Methods Human ARPE-19 cells were divided into control group, TGF-β1 treatment group, NAC intervention group and simple NAC group. Cells in the control group were not disposed, 10 ng/ml TGF-β1, 10 ng/ml TGF-β1+ 5 mmol/L NAC and 5 mmol/L NAC were used to interfere the ARPE-19 cells for 48 hours in the other 3 groups.The morphological changes of the cells were observed by phase contrast microscopy.Real-time quantitative PCR, Western blot, immunofluorescence and ELISA were used to detect the expression of transdifferentiation marker genes—α-smooth muscle actin (α-SMA), fibronectin and typeⅠcollagen.The expression level of intracellular ROS induced by TGF-β1 in the presence or absence of NAC was detected using the non-fluorescent probe DCFH-DA performed by flow cytometry.The phosphorylation level of p38MAPK, ERK1/2 and SAPK/JNK were detected by Western blot.The cell counting kit-8 (CCK-8) assay was used to investigate the effect of NAC on the viability of ARPE-19 cells. Results The expressions of α-SMA, fibronectin and type Ⅰ collagen mRNA in the TGF-β1 treatment group were significantly increased, and were (2.15±0.29), (9.54±1.08), (25.78±0.66) times higher than those of the control group, with significant differences between the two groups (all at P<0.05). The expressions of α-SMA, fibronectin and type Ⅰ collagen protein were significantly increased, and were (8.49±0.32), (2.53±0.69), (4.45±1.05) times higher than those of the control group, respectively, with significant differences between the two groups (all at P<0.05). The expressions of α-SMA, fibronectin and type Ⅰ collagen mRNA in the NAC intervention group were (66.70±12.57)%, (66.11±8.35)% and (33.19±6.90)% lower than those of the TGF-β1 treatment group, and the expressions of protein were (52.30±4.83)%, (55.03±2.58)% and (56.08±3.65)% lower than those of the TGF-β1 treatment group, respectively, with significant differences between the two groups (all at P<0.05). Flow cytometry results showed that the expression level of intracellular ROS in the TGF-β1 treatment group was (2.12±0.20) times higher than that of the control group, and the intracellular ROS in the NAC intervention group was (57.41±9.45)% lower than that of the TGF-β1 treatment group, with significant differences between the groups (both at P<0.01). Western blot results showed that the phosphorylation levels of p38MAPK, SAPK/JNK and ERK1/2 in the TGF-β1 treatment group were (9.18±1.00), (4.87±0.81) and (4.20±0.69) times higher than those of the control group, respectively, showing significant differences between the two groups (all at P<0.05). The phosphorylation level of p38MAPK, SAPK/JNK and ERK1/2 in the NAC intervention group were (48.16±14.82)%, (67.90±2.90)% and (74.52±4.00)% lower than those of the TGF-β1 treatment group, respectively, showing significant differences between the two groups (all at P<0.05). Conclusions NAC inhibits the TGF-β1-induced transdifferentiation of ARPE-19 cells possibly via suppressing the production of intracellular ROS and the phosphorylation of p38MAPK, SAPK/JNK and ERK1/2. Key words: N-acetylcysteine; Retinal pigment epithelial cell; Transforming growth factor-β1; Myofibroblast; Transdifferentiation; α-Smooth muscle actin

The surgical experience in recurrent retinal detachments after episcleral procedures

The two major surgical treatments of retinal detachments are episcleral and intravitreal procedures. There is no general consensus as to which procedure is the most appropriate for this complex disease. The aim of the study was to define differentiated surgical tactics, and to look at five year results of extrascleral surgical procedures based upon clinical and statistical analysis. . The results showed that primary rhegmatogenous retinal detachment with proliferative vitreoretinopathy (PVR) stage A is an indication for episcleral buckling if tears are located in the retinal periphery. If there are multiple tears, a concomitant macular hole or a large flap tear, the surgery may also include drainage of subretinal fluid, intravitreal air or gas injection, and avoidance of the use of cryotherapy. The causes for 19 recurrences that required subsequent vitreoretinal surgery are: PVR progression after surgery and as a consequence of it a retinal detachment recurrence, epiretinal fibrosis, subretinal proliferation.

Overexpression p21WAF1/CIP1 in suppressing retinal pigment epithelial cells and progression of proliferative vitreoretinopathy via inhibition CDK2 and cyclin E.

BackgroundP21 is one kind of cyclin-dependent kinase inhibitor that can prevent cells from going through the G1/S phase checkpoint and inhibit cell proliferation. Proliferative vitreoretinopathy (PVR) is a proliferative response in the eye. The aim of this study was to determine whether p21Waf1/Cip1 (p21) suppresses the proliferation and migration of retinal pigment epithelial (RPE) cells in vitro and controls PVR development in vivo.MethodsCell cycle analyses and transwell assays were conducted to assess cell proliferation characteristics and the migration ability of RPE cells after transfection with p21. Western blot and reverse-transcription polymerase chain reaction technologies were used to detect the expression of p21, CDK2 and cyclinE in RPE cells and rabbit retinal tissues. The impact of increasing p21 expression on PVR development was conducted by implantation of an adenovirus vector containing rabbit p21 (rAd-p21) in a PVR rabbit model. The prevalence of PVR and retinal detachment was determined by indirect ophthalmoscopy on days 3, 7, 14, and 21 after the injection of rAd-p21 into the vitreous. B scans and hematoxylin-eosin staining were employed to check rabbit retinas on day 21.ResultsCell cycle analyses and transwell assays showed that p21 inhibited the proliferation and migration of RPE cells. Increased expression of p21 was detected in cultured RPE cells and rabbit retinas after transfection with the p21 gene, whereas levels of CDK2 and cyclinE were decreased. The increase in p21 expression effectively suppressed the development of PVR in a rabbit model.ConclusionsThe increase in p21 expression in RPE cells not only inhibits the proliferation and migration of RPE cells in vitro, but also suppresses the development of PVR in vivo, which indicates its therapeutic potential in treating PVR.

Intravitreal Inflammation: From Benchside to Bedside 2013

The aim of this special issue is to describe the role of inflammation in biological mechanisms of long-known diseases, providing an up-to-date viewpoint for therapeutic approaches. The authors provide a comprehensive overview, with original and review articles, on the implications of inflammation in proliferative vitreous retinopathy, on diabetic retinopathy, and on the potential iatrogenic damage induced by sustained intravitreal anti-VEGF therapy. Despite the copious literature, the exact mechanism of idiopathic epiretinal membranes formation (iERM) is still unclear. M. Joshi et al. showed the importance of glial cells and hyalocytes in its pathogenesis. Such cellular constituents of iERMs in concert with cytokines, growth factors (TGF and NGF), and anomalous posterior vitreous detachment are responsible for the prognosis and potential growth of epiretinal traction. The proliferative vitreoretinopathy (PVR) is recognized as a common secondary severe inflammatory complication of retinal detachment. C. Azzolini et al. reported two possible pathways (VEGF-A, Otx2, p53, p63, and Otx1 and Otx3) of PVR. These genetic pathways could represent a novel target of therapy of PVR progression. PVR is an even more common complication in patients with open-globe injury. F. Morescalchi et al. describe the role of immune cells into the vitreous gel in penetrating ocular injury. The cells stimulate the production of growth factors and cytokines, in particular PDGFR-α, which seem to be crucial in the development of PVR and, therefore, considered another potential therapeutic target. In a prospective, nonrandomized, observational study, C. Costagliola et al. showed that tear fluid collection is a useful and noninvasive method for the finding of proliferative diabetic retinopathy. The authors reported that TNF-alpha concentrations increase in tears according to the severity of proliferative and nonproliferative diabetic retinopathy. F. Parmeggiani et al. demonstrated the evidence of a relationship between AMD-risk genotypes, immunoinflammatory endophenotypes, and the networks of acquired or epigenetic factors. The authors reported that proinflammatory effects secondary to chronic inflammation and heterogeneous types of oxidative stress induce degenerative damage to the photoreceptors and outer retinal-blood barrier. In particular, the carriers of CFH, ARMS2/HRTA1, and C2/CFB genotypes demonstrated high odd ratio values. A score, calculating a risk including genetic findings, could be useful to identify individual risk, in order to provide preventive treatments. The intraviteral inflammation can be also secondary to iatrogenic treatment as intravitreal anti-VEGF therapy or vitreoretinal surgery. S. Agrawal et al. compared the sterile inflammation among the different intravitreal anti-VEGF drugs currently used for neovascular diseases. The acute intraocular inflammation is more frequently associated with bevacizumab likely due to the less strict purification procedure of the drug. In most cases, the inflammation solves spontaneously and vision returns to baseline. A history of previous inflammation does not increase the risk with following intravitreal injections. Previous vitreoretinal surgery can also induce intravitreal inflammation responsible for cytotoxic macular oedema. V. Romano et al. reported that intravitreal inflammation may increase after pars plana vitrectomy due to tractional phenomena at vitreomacular interfaces or due to a vasogenic damage. The oedema is characterized by fluid accumulation in the parenchymatous retinal cells (intracellular oedema) or by extracellular fluid accumulation due to a blood-retinal barrier damage (extracellular oedema). The treatment of vitreous inflammation is still a challenging issue; however, there are multiple methods of systemic treatment including T-cell inhibitors/calcineurin, antimetabolites, corticosteroids, alkylating agents, inhibitors, and biologic agents. A. Jiang et al. showed that these drugs can be used either alone or together in order to control vitreous inflammation due to variety of etiologies, either infectious or noninfectious. A. Russo et al. demonstrated that the topical nonsteroidal anti-inflammatory drugs (NSAIDs) allow for greater drug penetration into the vitreous. Topical NSAIDs reduce the exudation secondary to age-related macular degeneration or diabetic maculopathy. NSAIDs are also considered a favorable adjunct together with anti-VEGF that could potentially reduce the number of anti-VEGF injections. Finally, the chronicity of certain inflammatory conditions limits the efficacy of locally administered drugs. J. Wang et al. reported the last updates on implantable devices and particulate delivery systems such as nanoparticles, microparticles, and liposomes. These topics are actually considered the research focus of biomedical engineering, pharmacology, and molecular biology. We wish that this special issue will provide helpful information to recognize the clinical features of vitreous inflammation, to understand the mechanism beyond, and to identify the latest treatments in the disease in which the vitreous is involved. Mario R. Romano John Christoforidis

Expression of IGFBP-6 in proliferative vitreoretinopathy rat models and its effects on retinal pigment epithelial-J cells

Proliferative vitreoretinopathy (PVR) is one of the most common causes for failed retinal detachment surgeries. The aim of the present study was to investigate the role of insulin‑like growth factor‑binding protein‑6 (IGFBP‑6) in PVR using rat models and its effects on retinal pigment epithelial‑J (RPE‑J) cells. PVR Wistar rat models were administered intravitreal injection of RPE‑J cells (1x106/5µl) combined with platelet‑rich plasma (1x107/5µl). The concentration of IGFBP‑6 in the vitreous and serum of rats was tested by an enzyme‑linked immunosorbent assay and the expression of IGFBP‑6 mRNA in the liver and retina of rats was determined by quantitative polymerase chain reaction (qPCR). The expression of IGFBP‑6 mRNA in the RPE‑J cells stimulated by vitreous or serum from PVR patients or normal volunteers was also determined by qPCR. The proliferation of RPE‑J cells was evaluated by the 3‑(4,5‑dimethylthiazol‑2‑yl)‑5‑(3‑carboxymethoxyphenyl)‑2‑(4‑sulfophenyl)‑2H‑tetrazolium, inner salt (MTS) method. The success rate of PVR rat model induction at the 8th week was 89.5% (34/38). The concentration of IGFBP‑6 in the vitreous and serum of PVR rats was significantly higher than that of the control group (P<0.05). The expression of IGFBP‑6 mRNA in the retina of PVR rats was also significantly higher compared with the control group (P<0.05). The vitreous from PVR patients and donors significantly stimulated the expression of IGFBP‑6 mRNA in the RPE‑J cells (P<0.05). IGFBP‑6 only inhibited IGF‑II‑stimulated proliferation but not the basal level of proliferation or the PDGF/VEGF‑stimulated RPE‑J cell proliferation. Thus, the trends and effects of IGFBP‑6 provide the possibility of PVR therapeutic targets, with the vitreous representing a significant environmental factor in the progression of PVR.

Comparison of Gene Expression Profile of Epiretinal Membranes Obtained from Eyes with Proliferative Vitreoretinopathy to That of Secondary Epiretinal Membranes

BackgroundProliferative vitreoretinopathy (PVR) is a destructive complication of retinal detachment and vitreoretinal surgery which can lead to severe vision reduction by tractional retinal detachments. The purpose of this study was to determine the gene expression profile of epiretinal membranes (ERMs) associated with a PVR (PVR-ERM) and to compare it to the expression profile of less-aggressive secondary ERMs.Methodology/Principal FindingsA PCR-amplified complementary DNA (cDNA) library was constructed using the RNAs isolated from ERMs obtained during vitrectomy. The sequence from the 5′ end was obtained for randomly selected clones and used to generate expressed sequence tags (ESTs). We obtained 1116 nonredundant clusters representing individual genes expressed in PVR-ERMs, and 799 clusters representing the genes expressed in secondary ERMs. The transcriptome of the PVR-ERMs was subdivided by functional subsets of genes related to metabolism, cell adhesion, cytoskeleton, signaling, and other functions, by FatiGo analysis. The genes highly expressed in PVR-ERMs were compared to those expressed in the secondary ERMs, and these were subdivided by cell adhesion, proliferation, and other functions. Querying 10 cell adhesion-related genes against the STRING database yielded 70 possible physical relationships to other genes/proteins, which included an additional 60 genes that were not detected in the PVR-ERM library. Of these, soluble CD44 and soluble vascular cellular adhesion molecule-1 were significantly increased in the vitreous of patients with PVR.Conclusions/SignificanceOur results support an earlier hypothesis that a PVR-ERM, even from genomic points of view, is an aberrant form of wound healing response. Genes preferentially expressed in PVR-ERMs may play an important role in the progression of PVR and could be served as therapeutic targets.

Expression of VEGF-A, Otx Homeobox and p53 Family Genes in Proliferative Vitreoretinopathy

Introduction. Proliferative vitreoretinopathy (PVR) is a severe inflammatory complication of retinal detachment. Pathological epiretinal membranes grow on the retina surface leading to contraction, and surgery fails in 5% to 10% of the cases. We evaluated the expression of VEGF-A, Otx1, Otx2, Otx3, and p53 family members from PVR specimens to correlate their role in inducing or preventing the pathology. Methods. Twelve retinal samples were taken from patients affected by PVR during therapeutic retinectomies in vitreoretinal surgery. Gene expression was evaluated using quantitative real-time reverse transcriptase PCR analysis and immunohistochemistry, using four healthy human retinae as control. Result. Controls showed basal expression of all genes. PVR samples showed little or no expression of Otx1 and variable expression of VEGF-A, Otx2, Otx3, p53, and p63 genes. Significant correlation was found among VEGF-A, Otx2, p53, and p63 and between Otx1 and Otx3. Conclusions. Otx homeobox, p53 family, and VEGF-A genes are expressed in PVR human retina. We individuated two possible pathways (VEGF-A, Otx2, p53, p63 and Otx1 and Otx3) involved in PVR progression that could influence in different manners the course of the pathology. Individuating the genetic pathways of PVR represents a novel approach to PVR therapies.