Annexin A2 promotes proliferative vitreoretinopathy in response to a macrophage inflammatory signal in mice.

Proliferative vitreoretinopathy is a vision-threatening response to penetrating ocular injury, for which there is no satisfactory treatment. In this disorder, retinal pigment epithelial cells, abandon their attachment to Bruch's membrane on the scleral side of the retina, transform into motile fibroblast-like cells, and migrate through the retinal wound to the vitreal surface of the retina, where they secrete membrane-forming proteins. Annexin A2 is a calcium-regulated protein that, in complex with S100A10, assembles plasmin-forming proteins at cell surfaces. Here, we show that, in proliferative vitreoretinopathy, recruitment of macrophages and directed migration of retinal pigment epithelial cells are annexin A2-dependent, and stimulated by macrophage inflammatory protein-1α/ß. These factors induce translocation of annexin A2 to the cell surface, thus enabling retinal pigment epithelial cell migration following injury; our studies reveal further that treatment of mice with intraocular antibody to either annexin A2 or macrophage inflammatory protein dampens the development of proliferative vitreoretinopathy in mice.

Silencing the long noncoding RNA MALAT1 inhibits vitreous-induced epithelial-mesenchymal transition in RPE cells by regulating the PDGFRs/AKT axis.

PURPOSE: Epithelial-mesenchymal transition (EMT) is a crucial pathological process that contributes to proliferative vitreoretinopathy (PVR), and research indicates that factors present in the vitreous that target cells play pivotal roles in regulating EMT. Experimental studies have confirmed that rabbit vitreous (RV) promotes EMT in human retinal pigment epithelial (RPE) cells. The long noncoding RNA (lncRNA) MALAT1 has been implicated in EMT in various diseases. Thus, this study aimed to investigate the involvement of lncRNA MALAT1 in vitreous-induced EMT in RPE cells. METHODS: MALAT1 was knocked down in ARPE-19 cells by short hairpin RNA (shRNA) transfection. Reverse transcription PCR (RT‒PCR) was used to evaluate MALAT1 expression, and Western blotting analysis was used to measure the expression of EMT-related proteins. Wound-healing, Transwell, and cell contraction assays were conducted to assess cell migration, invasion, and contraction, respectively. Additionally, cell proliferation was assessed using the CCK-8 assay, and cytoskeletal changes were examined by immunofluorescence. RESULTS: MALAT1 expression was significantly increased in ARPE-19 cells cultured with RV. Silencing MALAT1 effectively suppressed EMT and downregulated the associated factors snail1 and E-cadherin. Furthermore, silencing MALAT1 inhibited the RV-induced migration, invasion, proliferation, and contraction of ARPE-19 cells. Silencing MALAT1 also decreased RV-induced AKT and P53 phosphorylation. CONCLUSIONS: In conclusion, lncRNA MALAT1 participates in regulating vitreous-induced EMT in human RPE cells; these results provide new insight into the pathogenesis of PVR and offer a potential direction for the development of antiproliferative drugs.

Knockdown of HSPA13 Inhibits TGFß1-Induced Epithelial-Mesenchymal Transition of RPE by Suppressing the PI3K/Akt Signaling Pathway.

Purpose: This study aimed to explore the impact of HSPA13 on epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells and proliferative vitreoretinopathy (PVR) development, along with its associated molecular mechanisms. Methods: HSPA13 expression was evaluated in epiretinal membranes (ERMs) from patients with PVR using immunohistochemistry. The effects of HSPA13 knockdown on TGFß1-induced EMT in hESC-RPE cells were studied through quantitative PCR (qPCR), Western blot, and wound healing assays. Intracellular Ca2+ levels were measured using Fluo-8/AM incubation. A rat PVR model was induced by the intravitreal injection of RPE cells combined with platelet-rich plasma (PRP). RNA-seq was applied to study the molecular mechanism of HSPA13 knockdown-mediated EMT inhibition. Results: HSPA13 was found in human ERMs and its expression increased with TGFß1 treatment in hESC-RPE cells. Knockdown of HSPA13 inhibited TGFß1-induced EMT and migration. In the PVR rat model, HSPA13 was expressed in the ERMs and its knockdown in RPE cells reduced the development of PVR. Consistent with these observations, RNA-seq showed a global suppression of TGFß1-induced EMT and migration by shHSPA13 in RPE cells. Mechanistically, TGFß1 treatment increased intracellular Ca2+ levels, leading to an upregulation of HSPA13 expression. Downregulation of HSPA13 hindered the phosphorylation of PI3K/Akt in TGFß1-induced RPE cells. Conclusions: Our study revealed the involvement of HSPA13 in PVR development, as well as in TGFß1-induced EMT of RPE through the PI3K/Akt signaling pathway. Targeting HSPA13-related pathways involved in regulating EMT in RPE cells could serve as a novel therapeutic approach for patients with PVR.

Multi-omics profiling of retinal pigment epithelium reveals enhancer-driven activation of RANK-NFATc1 signaling in traumatic proliferative vitreoretinopathy.

During the progression of proliferative vitreoretinopathy (PVR) following ocular trauma, previously quiescent retinal pigment epithelial (RPE) cells transition into a state of rapid proliferation, migration, and secretion. The elusive molecular mechanisms behind these changes have hindered the development of effective pharmacological treatments, presenting a pressing clinical challenge. In this study, by monitoring the dynamic changes in chromatin accessibility and various histone modifications, we chart the comprehensive epigenetic landscape of RPE cells in male mice subjected to traumatic PVR. Coupled with transcriptomic analysis, we reveal a robust correlation between enhancer activation and the upregulation of the PVR-associated gene programs. Furthermore, by constructing transcription factor regulatory networks, we identify the aberrant activation of enhancer-driven RANK-NFATc1 pathway as PVR advanced. Importantly, we demonstrate that intraocular interventions, including nanomedicines inhibiting enhancer activity, gene therapies targeting NFATc1 and antibody therapeutics against RANK pathway, effectively mitigate PVR progression. Together, our findings elucidate the epigenetic basis underlying the activation of PVR-associated genes during RPE cell fate transitions and offer promising therapeutic avenues targeting epigenetic modulation and the RANK-NFATc1 axis for PVR management.

Genetic Linkage between CAPN5 and TYR Variants in the Context of Albinism and Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy Absence: A Case Report.

We present a case involving a patient whose clinical phenotype aligns with oculocutaneous albinism (OCA), yet exhibits a complex genotype primarily characterized by variants of unknown significance (VUS). An 11-year-old boy manifested iris hypopigmentation and translucency, pronounced photophobia, diminished visual acuity and stereopsis, nystagmus, reduced pigmentation of the retina, and foveal hypoplasia. Genetic testing was performed. A heterozygous missense VUS CAPN5 c.230A>G, p.(Gln77Arg), a heterozygous missense VUS TYR c.1307G>C, p.(Gly436Ala), and a heterozygous missense variant TYR c.1205G>A, p.(Arg402Gln) which was classified as a risk factor, were identified. We hypothesized that the TYR c.1307G>C, p.(Gly436Ala) variant is in genetic disequilibrium with the TYR c.1205G>A, p.(Arg402Gln) variant leading to deficient expression of melanogenic enzymes in retinal cells, resulting in the manifestation of mild OCA. Additionally, this study represents the case where we did not detect chiasmal misrouting in visual evoked potentials, nor did we observe a shift in the distribution of ganglion cell thickness from a temporal to a central position. Moreover, our patient's case supports the probable benign nature of the CAPN5 c.230A>G, p.(Gln77Arg) variant.

Bone morphogenetic protein 6 (BMP6) antagonises experimental proliferative vitreoretinopathy established by TGF-ß2 stimulation in retinal pigment epithelial cells through modulation of the p38 and JNK MAPK pathways.

The formation of the epiretinal fibrotic membrane by retinal pigment epithelial (RPE) cells is a primary pathological change for proliferative vitreoretinopathy (PVR). Bone morphogenetic protein 6 (BMP6) is an antifibrogenic factor in various cells. To date, it is still unknown whether BMP6 can interfere with the fibrogenesis of RPE cells during the progression of PVR. This work aimed to address the relationship between BMP6 and transforming growth factor-ß2 (TGF-ß2)-elicited fibrogenesis of RPE cells, an experimental model for studying PVR in vitro. The BMP6 level was down-regulated, while the TGF-ß2 level was up-regulated in the vitreous humor of PVR patients. The BMP6 level was down-regulated in human RPE cells challenged with TGF-ß2. The treatment of RPE cells with TGF-ß2 resulted in significant increases in proliferation, migration, epithelial-to-mesenchymal transition (EMT), and extracellular matrix (ECM) remodelling. These effects were found to be inhibited by the overexpression of BMP6 or exacerbated by the knockdown of BMP6. BMP6 overexpression reduced the phosphorylation of p38 and JNK in TGF-ß2-stimulated RPE cells, while BMP6 knockdown showed the opposite effects. The inhibition of p38 or JNK partially reversed the BMP6-silencing-induced promoting effects on TGF-ß2-elicited fibrogenesis in RPE cells. Taken together, BMP6 demonstrates the ability to counteract the proliferation, migration, EMT, and ECM remodelling of RPE cells induced by TGF-ß2. This is achieved through the regulation of the p38 and JNK MAPK pathways. These findings imply a potential connection between BMP6 and PVR, and highlight the potential application of BMP6 in therapeutic interventions for PVR.

Limited Hyperoxia-Induced Proliferative Retinopathy (LHIPR) as a Model of Retinal Fibrosis, Angiogenesis, and Inflammation.

The progression to fibrosis and traction in retinopathy of prematurity (ROP) and other ischemic retinopathies remains an important clinical and surgical challenge, necessitating a comprehensive understanding of its pathogenesis. Fibrosis is an unbalanced deposition of extracellular matrix components responsible for scar tissue formation with consequent tissue and organ impairment. Together with retinal traction, it is among the main causes of retinal detachment and vision loss. We capitalize on the Limited Hyperoxia Induced Retinopathy (LHIPR) model, as it reflects the more advanced pathological phenotypes seen in ROP and other ischemic retinopathies. To model LHIPR, we exposed wild-type C57Bl/6J mouse pups to 65% oxygen from P0 to P7. Then, the pups were returned to room air to recover until later endpoints. We performed histological and molecular analysis to evaluate fibrosis progression, angiogenesis, and inflammation at several time points, from 1.5 months to 9 months. In addition, we performed in vivo retinal imaging by optical coherence tomography (OCT) or OCT Angiography (OCTA) to follow the fibrovascular progression in vivo. Although the retinal morphology was relatively preserved, we found a progressive increase in preretinal fibrogenesis over time, up to 9 months of age. We also detected blood vessels in the preretinal space as well as an active inflammatory process, altogether mimicking advanced preretinal fibrovascular disease in humans.

Autosomal dominant neovascular inflammatory vitreoretinopathy with CAPN5 c.731T > C gene mutation; clinical management of a family cohort and review of the literature.

BACKGROUND: To report a cohort of patients with clinically and genetically diagnosed autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) and showcase the spectrum of the disease utilizing multimodal imaging and genetic testing. Additionally, the utility of multimodal imaging in guiding treatment will also be illustrated. MATERIALS/METHODS: Five patients from a single-family pedigree in Ohio with clinical signs of ADNIV were evaluated. Medical history, family history, and complete ocular examinations were obtained during regular clinic visits. Multimodal imaging including ocular coherence tomography, fluorescein angiography, wide-field fundus photographs, and Humphrey visual field testing was obtained for all five patients. Additionally, genetic testing for the Calpain-5 (CAPN5) gene was conducted on all patients. RESULTS: All five patients were noted to have a CAPN5 c.731T > C (p.L244P) mutation on genetic testing. Using multimodal imaging to supplement the clinical examination, pathologic changes such as retinal vascular inflammation, macular edema, and tractional retinal membranes were well illustrated and monitored over time. This allowed for earlier intervention when appropriate such as with intraocular steroid or systemic anti-inflammatory treatments. CONCLUSION: Phenotypic presentation varied among patients in this series, but is consistent with the spectrum of pathologic changes previously described in patients with other CAPN5 gene mutations. Monitoring of patients with ADNIV utilizing multimodal imaging can help better assess progression of this disease and guide treatment decisions. Additionally, increased genetic testing in patients with inherited retinal diseases may reveal novel gene mutations that could serve as potential targets for future genetic treatment regimens.

Mechanism underlying Müller cell pyroptosis and its role in the development of proliferative vitreoretinopathy.

OBJECTIVES: To explore the mechanism underlying Müller Cell Pyroptosis (MCP) and its role in the development of Proliferative Vitreoretinopathy (PVR). METHOD: The expression of pyroptosis-related factors, namely, cysteinyl aspartate-specific proteinase (caspase-1), interleukin (IL)-1ß, IL-18, and Gasdermin D (GSDMD), was detected by quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) and western blotting at the mRNA and protein levels, respectively, in retinal tissues. Müller and spontaneously Arising Retinal Pigment Epithelia (ARPE)-19 primary cells with GSDMD overexpression or knockdown were cultivated. Western blotting was used to detect the levels of the following pyroptosis-related factors in retinal tissues: caspase-1, IL-1ß, IL-18, and GSDMD. Through Cell Adhesion (CA) experiments, the changes in ARPE-19 CA in each group were observed. The migration and invasion of ARPE-19 cells were measured using the Transwell assay. The proliferation of ARPE-19 cells was measured with a Cell Counting Kit 8 (CCK-8) assay. Finally, the expression of the cytokines IL-1ß and IL-18 in the ARPE-19 cell culture medium was detected using the Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: Compared with the surrounding normal tissues, the expression of caspase-1, IL-1ß, IL-18, and GSDMD at the protein and mRNA levels in the retinal proliferative membrane samples of the patients decreased significantly (p < 0.05). MCP significantly enhanced ARPE-19 CA, migration and invasion, proliferation, and cytokine expression (p < 0.05). CONCLUSIONS: MCP can promote the development of PVR lesions.

Palmitic acid-induced microRNA-143-5p expression promotes the epithelial-mesenchymal transition of retinal pigment epithelium via negatively regulating JDP2.

BACKGROUND: The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is the most crucial step in the etiopathogenesis of proliferative vitreoretinopathy. This study aimed to investigate the role of miR-143-5p in the EMT of RPE cells induced by palmitic acid (PA). METHODS: ARPE-19 cells were treated with PA to induce EMT, followed by E-cadherin and α-smooth muscle actin (α-SMA) expression and the microRNA expression profile analyses. Subsequently, miR-143-5p mimics/inhibitors, and plasmids expressing its predicted target gene c-JUN-dimerization protein 2 (JDP2), were transfected in ARPE-19 cells using lipofectamine 3000, and followed by PA treatment. Their impacts on EMT were explored using wound healing and Western blot assays. Additionally, miR-143-5p mimics and JDP2-expressing plasmid were co-transfected into ARPE-19 cells and treated with PA to explore whether PA induced EMT of ARPE-19 cells via the miR-143-5p/JDP2 axis. RESULTS: PA decreased E-cadherin expression and increased those of α-SMA and miR-143-5p. Inhibiting miR-143-5p suppressed the migration of ARPE-19 cells and altered the expressions of E-cadherin and α-SMA. However, additional PA treatment attenuated these alterations. JDP2 was a target of miR-143-5p. Overexpression of JDP2 inhibited the EMT of ARPE-19 cells, resulting in α-SMA downregulation and E-cadherin upregulation, which were reversed by additional PA treatment via inhibiting JDP2 expression. Overexpression of miR-143-5p reversed the effect of JDP2 on the EMT of ARPE-19 cells and additional PA treatment markedly enhanced the effect of miR-143-5p mimics. CONCLUSION: PA promotes EMT of ARPE-19 cells via regulating the miR-143-5p/JDP2 axis, and these findings provide significant insights into the potential targeting of this axis to treat proliferative vitreoretinopathy.

Impaired activity and membrane association of most calpain-5 mutants causal for neovascular inflammatory vitreoretinopathy.

Neovascular inflammatory vitreoretinopathy (NIV) is a rare eye disease that ultimately leads to complete blindness and is caused by mutations in the gene encoding calpain-5 (CAPN5), with six pathogenic mutations identified. In transfected SH-SY5Y cells, five of the mutations resulted in decreased membrane association, diminished S-acylation, and reduced calcium-induced autoproteolysis of CAPN5. CAPN5 proteolysis of the autoimmune regulator AIRE was impacted by several NIV mutations. R243, L244, K250 and the adjacent V249 are on ß-strands in the protease core 2 domain. Conformational changes induced by Ca2+binding result in these ß-strands forming a ß-sheet and a hydrophobic pocket which docks W286 side chain away from the catalytic cleft, enabling calpain activation based on comparison with the Ca2+-bound CAPN1 protease core. The pathologic variants R243L, L244P, K250N, and R289W are predicted to disrupt the ß-strands, ß-sheet, and hydrophobic pocket, impairing calpain activation. The mechanism by which these variants impair membrane association is unclear. G376S impacts a conserved residue in the CBSW domain and is predicted to disrupt a loop containing acidic residues which may contribute to membrane binding. G267S did not impair membrane association and resulted in a slight but significant increase in autoproteolytic and proteolytic activity. However, G267S is also identified in individuals without NIV. Combined with the autosomal dominant pattern of NIV inheritance and evidence that CAPN5 may dimerize, the results are consistent with a dominant negative mechanism for the five pathogenic variants which resulted in impaired CAPN5 activity and membrane association and a gain-of-function for the G267S variant.

DAPL1 prevents epithelial-mesenchymal transition in the retinal pigment epithelium and experimental proliferative vitreoretinopathy.

Epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a hallmark of the pathogenesis of proliferative vitreoretinopathy (PVR) that can lead to severe vision loss. Nevertheless, the precise regulatory mechanisms underlying the pathogenesis of PVR remain largely unknown. Here, we show that the expression of death-associated protein-like 1 (DAPL1) is downregulated in PVR membranes and that DAPL1 deficiency promotes EMT in RPE cells in mice. In fact, adeno-associated virus (AAV)-mediated DAPL1 overexpression in RPE cells of Dapl1-deficient mice inhibited EMT in physiological and retinal-detachment states. In a rabbit model of PVR, ARPE-19 cells overexpressing DAPL1 showed reduced ability to induce experimental PVR, and AAV-mediated DAPL1 delivery attenuated the severity of experimental PVR. Furthermore, a mechanistic study revealed that DAPL1 promotes P21 phosphorylation and its stabilization partially through NFκB (RelA) in RPE cells, whereas the knockdown of P21 led to neutralizing effects on DAPL1-dependent EMT inhibition and enhanced the severity of experimental PVR. These results suggest that DAPL1 acts as a novel suppressor of RPE-EMT and has an important role in antagonizing the pathogenesis of experimental PVR. Hence, this finding has implications for understanding the mechanism of and potential therapeutic applications for PVR.

Vitreous humor proteome: unraveling the molecular mechanisms underlying proliferative and neovascular vitreoretinal diseases.

Proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR), and neovascular age-related macular degeneration (nAMD) are among the leading causes of blindness. Due to the multifactorial nature of these vitreoretinal diseases, omics approaches are essential for a deeper understanding of the pathophysiologic processes underlying the evolution to a proliferative or neovascular etiology, in which patients suffer from an abrupt loss of vision. For many years, it was thought that the function of the vitreous was merely structural, supporting and protecting the surrounding ocular tissues. Proteomics studies proved that vitreous is more complex and biologically active than initially thought, and its changes reflect the physiological and pathological state of the eye. The vitreous is the scenario of a complex interplay between inflammation, fibrosis, oxidative stress, neurodegeneration, and extracellular matrix remodeling. Vitreous proteome not only reflects the pathological events that occur in the retina, but the changes in the vitreous itself play a central role in the onset and progression of vitreoretinal diseases. Therefore, this review offers an overview of the studies on the vitreous proteome that could help to elucidate some of the pathological mechanisms underlying proliferative and/or neovascular vitreoretinal diseases and to find new potential pharmaceutical targets.

Development of Proliferative Vitreoretinopathy Is Attenuated by Chicken Ovalbumin Upstream Promoter Transcriptional Factor 1 Via Inhibiting Epithelial-Mesenchymal Transition.

Proliferative vitreoretinopathy (PVR) is an intractable condition after rhegmatogenous retinal detachment (RD), which is the primary cause of failure in retinal reattachment surgery. This study aimed to investigate the effects of chicken ovalbumin upstream promoter transcriptional factor 1 (COUP-TF1) in the development of proliferative vitreoretinopathy (PVR) both in vitro and in vivo. Adult retinal pigment epithelium cell line was used for in-vitro experiments. Immunocytochemistry assay, real-time quantitative polymerase chain reaction, and Western blot were used to measure the expression of COUP-TF1, alpha-smooth muscle actin (α-SMA), and E-cadherin. Epithelial-mesenchymal transition (EMT) was observed through cell counting kit-8 assay, wound healing tests, and the expression changes of related proteins. PVR rabbit models were established and evaluated by the images of fundus and vitreous cavity, pathological sections, and COUP-TF1 expression. As shown by our results, the proliferation and migration of the COUP-TF1 knockdown cells were reduced compared with the control cells with or without transforming growth factor-ß1 (TGF-ß1) treatment. After TGF-ß1 treatment, α-SMA expression was upregulated in ARPE-19 cells but kept the same in COUP-TF1 knockdown cells. E-cadherin expression was down-regulated in all the groups but the extent of the decrease in COUP-TF1 knockdown cells was smaller. EMT was attenuated in ARPE-19 cells after COUP-TF1 was knocked down. In the in-vivo experiment, PVR severity was attenuated and the retinal detachment rate decreased on the 14th and 28th day in COUP-TF1 knockdown group. In conclusion, COUP-TF1 is related to the development of PVR, and COUP-TF1 knockdown attenuates the progression of PVR. This suggests that COUP-TF1 can be a promising candidate for the treatment of PVR.

Dopamine receptor signaling regulates fibrotic activation of retinal pigmented epithelial cells.

Retinal pigmented epithelial (RPE) cells play an important role in retinal fibrotic diseases such as proliferative vitreoretinopathy (PVR). The purpose of this study was to elucidate the involvement of dopamine receptor signaling in regulating the fibrotic activation of RPE cells. Dopamine receptor expression, the effect of dopamine on fibrotic activity, and dopamine production were measured in the human RPE cell line ARPE-19. The fibrotic activation of RPE cells was evaluated in response to treatments with selective dopamine receptor agonists and antagonists by measuring gene expression, migration, proliferation, and fibronectin deposition. DRD2 and DRD5 are the dominant dopaminergic receptors expressed in ARPE-19 cells and TGF-ß stimulation enhances the autocrine release of dopamine, which we show further exasperates fibrotic activation. Finally, treatment with D2 dopamine receptor antagonists or D5 dopamine receptor agonists inhibits profibrotic gene expression, migration, proliferation, and fibronectin deposition and thus may serve as effective mechanisms for treating retinal fibrosis including PVR.

IL-33 enhances Jagged1 mediated NOTCH1 intracellular domain (NICD) deubiquitination and pathological angiogenesis in proliferative retinopathy.

Pathological retinal neovascularization (NV) is a clinical manifestation of various proliferative retinopathies, and treatment of NV using anti-VEGF therapies is not selective, as it also impairs normal retinal vascular growth and function. Here, we show that genetic deletion or siRNA-mediated downregulation of IL-33 reduces pathological NV in a murine model of oxygen-induced retinopathy (OIR) with no effect on the normal retinal repair. Furthermore, our fluorescent activated cell sorting (FACS) data reveals that the increase in IL-33 expression is in endothelial cells (ECs) of the hypoxic retina and conditional genetic deletion of IL-33 in retinal ECs reduces pathological NV. In vitro studies using human retinal microvascular endothelial cells (HRMVECs) show that IL-33 induces sprouting angiogenesis and requires NFkappaB-mediated Jagged1 expression and Notch1 activation. Our data also suggest that IL-33 enhances de-ubiquitination and stabilization of Notch1 intracellular domain via its interaction with BRCA1-associated protein 1 (BAP1) and Numb in HRMVECs and a murine model of OIR.

Limited hyperoxia-induced proliferative retinopathy: A model of persistent retinal vascular dysfunction, preretinal fibrosis and hyaloidal vascular reprogramming for retinal rescue.

BACKGROUND: Retinopathy of prematurity (ROP) remains the leading cause for blindness in children. Limited hyperoxia induced proliferative retinopathy (L-HIPR) was recently introduced as a potential animal model for ROP and persistent fetal vasculature; however, the detailed pathological changes remain unclear. METHODS: To model L-HIPR, we placed C57BL/6J mice in 65% oxygen from birth to post-natal day 7 (P7). We examined eyes at intervals between P12 and P30. Retinal morphometry, thickness, and preretinal fibrosis were quantified at different time points on histological sections stained with hematoxylin and eosin (H&E) and Masson Trichrome, respectively. Vascular development, angiogenesis, inflammation, and pericyte coverage were analyzed using immunohistochemistry staining in retinal flat mounts and cross sections. RESULTS: In L-HIPR, the hyaloidal vessels persisted until the latest time point in this study, P30 and began to invaginate the peripheral then central retina starting at P12. Central retinal distortion was noted beginning at P17, while the peripheral retina demonstrated a trend of thinning from P12 to P30. We found that L-HIPR was associated with delayed and abnormal retinal vascular development with subsequent retinal inflammation, pericyte loss and preretinal fibrosis. CONCLUSION: Our study presents a detailed analysis of the L-HIPR animal model demonstrating vitreoretinal pathologic changes, preretinal fibrosis and persistent hyaloidal vessels into adulthood. Based on our findings, we suggest that the persistence and peculiar stepwise migration of the hyaloidal vessels into the retina may provide a potential rescue mechanism for inner retinal development that deserves further study.

Postoperative proliferative vitreoretinopathy development is linked to vitreal CXCL5 concentrations.

The specific changes linked to de novo development of postoperative PVR have remained elusive and were the object of the underlying study. Vitreous fluid (VF) was obtained at the beginning of vitrectomy from 65 eyes that underwent vitrectomy for primary rhegmatogenous retinal detachment (RRD) without preoperative PVR. Eyes developing postoperative PVR within 6 months after re-attachment surgery were compared to those which did not regarding the preoperative concentrations of 43 cytokines and chemokines in the VF, using multiplex beads analysis. For all comparisons Holm's correction was applied in order to control for multiple comparisons. Twelve out of 65 eyes (18.5%) developed PVR postoperatively. While 12 of the chemokines and cytokines presented concentration differences on a statistical level of p < 0.05 (CXCL5, CCL11, CCL24, CCL26, GM-CSF, IFN-γ, CCL8, CCL7, MIF, MIG/CXCL9, CCL19, and CCL25), CXCL5 was the only cytokine with sufficiently robust difference in its VF concentrations to achieve significance in eyes developing postoperative PVR compared to eyes without PVR. CXCL5 may represent a potent biomarker for the de novo development of postoperative PVR. In line with its pathophysiological role in the development of PVR, it might serve as a basis for the development of urgently needed preventive options.

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

This study was aim to investigate whether the progression of proliferative vitreoretinopathy (PVR) depended on the activation of Yes-associated protein (YAP) and the subsequent epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cell. The effect of YAP activation on retinal fibrosis in a PVR mouse model and in human ARPE-19 cells in vitro was studied. After treated with transforming growth factor-ß2(TGF-ß2), the expressions of fibrogenic molecules, YAP activation and the TGF-ß2-Smad signalling pathway in ARPE-19 cells were detected by Western blot and immunocytochemical analyses. The effect of YAP on change in fibrosis and EMT was tested by knockdown experiment using verteporfin (YAP inhibitor). YAP was upregulated in the PVR mouse model and during TGF-ß2-induced RPE cell EMT. In an in vivo study, verteporfin attenuated PVR progression in a mouse model. Additionally, YAP knockdown retained phenotype of RPE cells and ameliorated TGF-ß2-induced migration, gel contraction and EMT in vitro. YAP knockdown inhibited the TGF-ß2-induced upregulation of connective tissue growth factor (CTGF), smooth muscle actin (SMA-α) and fibronectin. YAP was essential for the TGF-ß2-induced nuclear translocation and phosphorylation of Smad2/3. Our work provides direct evidence that YAP is an essential regulator of EMT and profibrotic responses in PVR and indicates that YAP inhibition could be a potential target in PVR therapeutic intervention.

Repression of Smad4 by MicroRNA-1285 moderates TGF-ß-induced epithelial-mesenchymal transition in proliferative vitreoretinopathy.

The purpose of this study was to assess whether microRNA (miR)-1285 can suppress the epithelial-mesenchymal transition (EMT) in retinal pigment epithelial cells. Expression of miR-1285 was evaluated using quantitative real-time polymerase chain reaction (RT-qPCR). The features of EMT were assessed using Western blotting, immunocytochemical staining, scratch wound healing tests, modified Boyden chamber assay, and collagen gel contraction assay. A rabbit model of proliferative vitreoretinopathy (PVR) was used for in vivo testing, which involved the induction of PVR by injection of transfected ARPE cells into the vitreous chamber. Luciferase reporter assay was performed to identify the putative target of miR-1285. The expression of miR-1285 was downregulated in ARPE-19 cells treated with transforming growth factor (TGF)-ß. Overexpression of miR-1285 led to upregulation of zonula occludens-1, downregulation of α-smooth muscle actin and vimentin, cell migration and cell contractility-all EMT features-in the TGF-ß2-treated ARPE-19 cells. The reporter assay indicated that the 3' untranslated region of Smad4 was the direct target of miR1285. PVR progression was alleviated in the miR-1285 transfected rabbits. In conclusion, overexpression of miR-1285 attenuates TGF-ß2-induced EMT in a rabbit model of PVR, and the effect of miR-1285 in PVR is dependent on Smad4. Further research is warranted to develop a feasible therapeutic approach for the prevention and treatment of PVR.