SUMOylation of GMFB regulates its stability and function in retinal pigment epithelial cells under hyperglycemia.

BACKGROUND: Glia maturation factor beta (GMFB) is a growth and differentiation factor that acts as an intracellular regulator of signal transduction pathways. The small ubiquitin-related modifier (SUMO) modification, SUMOylation, is a posttranslational modification (PTM) that plays a key role in protein subcellular localization, stability, transcription, and enzymatic activity. Recent studies have highlighted the importance of SUMOylation in the inflammation and progression of numerous diseases. However, the relationship between GMFB and SUMOylation is unclear. RESULTS: Here, we report for the first time that GMFB and SUMO1 are markedly increased in retinal pigment epithelial (RPE) cells at the early stage of diabetes mellitus (DM) under hyperglycemia. The GMFΒ protein could be mono-SUMOylated by SUMO1 at the K20, K35, K58 or K97 sites. SUMOylation of GMFB led to its increased protein stability and subcellular translocation. Furthermore, deSUMOylation of GMFΒ downregulates multiple signaling pathways, including the Jak-STAT signaling pathway, p38 pathway and NF-kappa B signaling pathway. CONCLUSIONS: This work provides novel insight into the role of SUMOylated GMFB in RPE cells and provides a novel therapeutic target for diabetic retinopathy (DR).

A comprehensive stem cell laboratory module with blended learning for medical students at Tongji University.

The laboratory practice "Primary culture and directional differentiation of rat bone marrow mesenchymal stem cells (BMSCs)" is part of a required course for sophomore medical students at Tongji university, which has been conducted since 2012. Blended learning has been widely applied in medical courses. Based on a student-centered teaching philosophy, we reconstructed a comprehensive stem cell laboratory module with blended learning in 2021, aiming to facilitate students in enhancing their understanding of the multi-lineage differentiation potential of stem cells and improve their experimental skills, self-directed learning ability, and innovative thinking. First, we constructed in-depth online study resources, including videos demonstrating laboratory procedures, a PowerPoint slide deck, and published literature on student self-learning before class. In class, students performed a primary culture of BMSCs, freely chose among adipogenic, osteogenic, or chondrogenic differentiation, and used cytochemical or immunofluorescence staining for identification. After class, the extracurricular part involved performing quantitative polymerase chain reaction to examine the expression of multi-lineage differentiation marker genes, which was designed as an elective. After 2 years of practice, positive feedback was obtained from both students and faculty members who achieved, the learning goal as expected. The reconstructed stem cell laboratory module provides comprehensive practice opportunities for students. Students have a better understanding of BMSC at the molecular, cellular, and functional levels and have improved their experimental skills, which forms a basis for scientific research for medical students. Introducing blended learning into other medical laboratory practices thus seems valuable.

GMFB/AKT/TGF-ß3 in Müller cells mediated early retinal degeneration in a streptozotocin-induced rat diabetes model.

Retinal degeneration, characterized by Müller cell gliosis and photoreceptor apoptosis, is considered an early event in diabetic retinopathy (DR). Our previous study proposed that GMFB may mediate diabetic retinal degeneration. This study identified GMFB as a sensitive and functional gliosis marker for DR. Compared to the wild type (WT) group, Gmfb knockout (KO) significantly improved visual function, attenuated gliosis, reduced the apoptosis of neurons, and decreased the mRNA levels of tumor necrosis factor α (Tnf-α) and interleukin-1ß (Il-1ß) in diabetic retinas. Tgf-ß3 was enriched by hub genes using RNA sequencing in primary WT and KO Müller cells. Gmfb KO significantly upregulated the transforming growth factor (TGF)-ß3 protein level via the AKT pathway. The protective effect of TGF-ß3 in the vitreous resulted in significantly improved visual function and decreased the number of apoptotic cells in the diabetic retina. The protection of Gmfb KO in primary Müller cells against high glucose (HG)-induced photoreceptor apoptosis was partially counteracted by TGF-ß3 antibody and administration of TGFBR1/2 inhibitors. Nuclear receptor subfamily 3 group C member 1 (NR3C1) binds to the promoter region of Gmfb and regulates Gmfb mRNA at the transcriptional level. NR3C1 was increased in the retinas of early diabetic rats but decreased in the retinas of late diabetic rats. N'-[(1E)-(3-Methoxyphenyl)Methylene]-3-Methyl-1H-Pyrazole-5-Carbohydrazide (DS-5) was identified as an inhibitor of GMFB, having a protective role in DR. We demonstrated that GMFB/AKT/TGF-ß3 mediated early diabetic retinal degeneration in diabetic rats. This study provides a novel therapeutic strategy for treating retinal degeneration in patients with DR.

Subconjunctival injection of human umbilical cord mesenchymal stem cells alleviates experimental allergic conjunctivitis via regulating T cell response.

BACKGROUND: T helper 2 (Th2) cells are thought to play critical roles in allergic conjunctivitis (AC). They release inflammatory cytokines to promote an allergic response in AC. Due to individual heterogeneity and long-term chronic management, current therapies do not always effectively control AC. Mesenchymal stem cells (MSCs) have been shown to be effective in treating allergy-related disorders, but it is unclear how exactly the Th2-mediated allergic response is attenuated. This study aims to elucidate the therapeutic effect and mechanism of the human umbilical cord MSCs (hUCMSCs) in a mouse model of experimental AC (EAC). METHODS: A mouse EAC model was established by inoculating short ragweed (SRW) pollen. After the SRW pollen challenge, the mice received a single subconjunctival or tail vein injection of 2 × 106 hUCMSCs, or subconjunctival injection of hUCMSCs conditioned medium (hUCMSC-CM), and dexamethasone eye drops was used as positive control; subsequent scratching behavior and clinical symptoms were assessed. Immunostaining and flow cytometry were carried out to show allergic reactions and the activation of CD4 + T cell subsets in the conjunctiva and cervical lymph nodes (CLNs). Gene expression was determined by RNA-seq and further verified by qRT-PCR and Western blot. Co-culture assays were performed to explore the regulatory role of hUCMSCs in the differentiation of CD4 + naive T cells (Th0) into Th2 cells. RESULTS: Subconjunctival administration of hUCMSCs resulted in fewer instances of scratching and lower inflammation scores in EAC mice compared to the tail vein delivery, hUCMSC-CM and control groups. Subconjunctival administration of hUCMSCs reduced the number of activated mast cells and infiltrated eosinophils in the conjunctiva, as well as decreased the number of Th2 cells in CLNs. After pretreatment with EAC mouse serum in vitro to mimic the in vivo milieu, hUCMSCs were able to inhibit the differentiation of Th0 into Th2 cells. Further evidence demonstrated that repression of Th2 cell differentiation by hUCMSCs is mediated by CRISPLD2 through downregulation of STAT6 phosphorylation. Additionally, hUMCSCs were able to promote the differentiation of Th0 cells into regulatory T cells in CLNs of EAC mice. CONCLUSIONS: Subconjunctival injection of hUCMSCs suppressed the Th2-allergic response and alleviated clinical symptoms. This study provides not only a potential therapeutic target for the treatment of AC but also other T cell-mediated diseases.

Glia maturation factor beta deficiency protects against diabetic osteoporosis by suppressing osteoclast hyperactivity.

Excessive osteoclast activation, which depends on dramatic changes in actin dynamics, causes osteoporosis (OP). The molecular mechanism of osteoclast activation in OP related to type 1 diabetes (T1D) remains unclear. Glia maturation factor beta (GMFB) is considered a growth and differentiation factor for both glia and neurons. Here, we demonstrated that Gmfb deficiency effectively ameliorated the phenotype of T1D-OP in rats by inhibiting osteoclast hyperactivity. In vitro assays showed that GMFB participated in osteoclast activation rather than proliferation. Gmfb deficiency did not affect osteoclast sealing zone (SZ) formation but effectively decreased the SZ area by decreasing actin depolymerization. When GMFB was overexpressed in Gmfb-deficient osteoclasts, the size of the SZ area was enlarged in a dose-dependent manner. Moreover, decreased actin depolymerization led to a decrease in nuclear G-actin, which activated MKL1/SRF-dependent gene transcription. We found that pro-osteoclastogenic factors (Mmp9 and Mmp14) were downregulated, while anti-osteoclastogenic factors (Cftr and Fhl2) were upregulated in Gmfb KO osteoclasts. A GMFB inhibitor, DS-30, targeting the binding site of GMFB and Arp2/3, was obtained. Biocore analysis revealed a high affinity between DS-30 and GMFB in a dose-dependent manner. As expected, DS-30 strongly suppressed osteoclast hyperactivity in vivo and in vitro. In conclusion, our work identified a new therapeutic strategy for T1D-OP treatment. The discovery of GMFB inhibitors will contribute to translational research on T1D-OP.

Aquaporin 11 alleviates retinal Müller intracellular edema through water efflux in diabetic retinopathy.

Retinal Müller glial dysfunction and intracellular edema are important mechanisms leading to diabetic macular edema (DME). Aquaporin 11 (AQP11) is primarily expressed in Müller glia with unclear functions. This study aims to explore the role of AQP11 in the pathogenesis of intracellular edema of Müller glia in diabetic retinopathy (DR). Here, we found that AQP11 expression, primarily located at the endfeet of Müller glia, was down-regulated with diabetes progression, accompanied by intracellular edema, which was alleviated by intravitreal injection of lentivirus-mediated AQP11 overexpression. Similarly, intracellular edema of hypoxia-treated rat Müller cell line (rMC-1) was aggravated by AQP11 inhibition, while attenuated by AQP11 overexpression, accompanied by enhanced function in glutamate metabolism and reduced cell death. The down-regulation of AQP11 was also verified in the Müller glia from the epiretinal membranes (ERMs) of proliferative DR (PDR) patients. Mechanistically, down-regulation of AQP11 in DR was mediated by the HIF-1α-dependent and independent miRNA-AQP11 axis. Overall, we deciphered the AQP11 down-regulation, mediated by miRNA-AQP11 axis, resulted in Müller drainage dysfunction and subsequent intracellular edema in DR, which was partially reversed by AQP11 overexpression. Our findings propose a novel mechanism for the pathogenesis of DME, thus targeting AQP11 regulation provides a new therapeutic strategy for DME.

Direct conversion of human umbilical cord mesenchymal stem cells into retinal pigment epithelial cells for treatment of retinal degeneration.

Age-related macular degeneration (AMD) is a major vision-threatening disease. Although mesenchymal stem cells (MSCs) exhibit beneficial neural protective effects, their limited differentiation capacity in vivo attenuates their therapeutic function. Therefore, the differentiation of MSCs into retinal pigment epithelial (RPE) cells in vitro and their subsequent transplantation into the subretinal space is expected to improve the outcome of cell therapy. Here, we transdifferentiated human umbilical cord MSCs (hUCMSCs) into induced RPE (iRPE) cells using a cocktail of five transcription factors (TFs): CRX, NR2E1, C-MYC, LHX2, and SIX6. iRPE cells exhibited RPE specific properties, including phagocytic ability, epithelial polarity, and gene expression profile. In addition, high expression of PTPN13 in iRPE cells endows them with an epithelial-to-mesenchymal transition (EMT)-resistant capacity through dephosphorylating syntenin1, and subsequently promoting the internalization and degradation of transforming growth factor-ß receptors. After grafting into the subretinal space of the sodium iodate-induced rat AMD model, iRPE cells demonstrated a better therapeutic function than hUCMSCs. These results suggest that hUCMSC-derived iRPE cells may be promising candidates to reverse AMD pathophysiology.

Elimination of senescent cells inhibits epithelial-mesenchymal transition of retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is one of the most common leading causes of irreversible blindness, and there is no effective treatment for it. It has been reported that aging is the greatest risk factor for AMD, and epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells plays an important role in the pathogenesis of AMD. To clarify the relationship between senescence and EMT in RPE cells, we used the replicative senescence model, H2O2- and/or Nutlin3a-induced senescence model, and low-density and/or TGF-ß-induced EMT model to detect the expression of senescence-, RPE- and EMT-related genes, and assessed the motility of cells by using a scratch wound migration assay. The results showed that replicative senescence of RPE cells was accompanied by increased expression of EMT markers. However, senescent RPE cells themselves did not undergo EMT, as the H2O2and Nutlin3a treated cells showed no increase in EMT characteristics, including unchanged or decreased expression of EMT markers and decreased motility. Furthermore, conditioned medium (CM) from senescent cells induced EMT in presenescent RPE cells, and EMT accelerated the process of senescence. Importantly, dasatinib plus quercetin, which selectively eliminates senescent cells, inhibited low-density-induced EMT in RPE cells. These findings provide a better understanding of the interconnection between senescence and EMT in RPE cells. Removal of senescent cells by certain methods such as senolytics, might be a promising potential approach to prevent or delay the progression of RPE-EMT-related retinal diseases such as AMD.

Glia Maturation Factor ß as a Novel Independent Prognostic Biomarker and Potential Therapeutic Target of Kidney Renal Clear Cell Carcinoma.

Kidney renal clear cell carcinoma (KIRC) has the highest mortality rate and potential for invasion among renal cancers. The diagnosis and treatment of KIRC are becoming challenging because of its diverse pathogenic mechanisms. Glia (GMFB) is a highly conserved growth and differentiation factor for glia cells and neurons, and it is closely associated with neurodegenerative diseases. However, its role in KIRC remains unknown. The present study integrated bioinformatics approaches with suitable meta-analyses to determine the position of GMFB in KIRC. There was a significant decrease in Gmfb expression in KIRC kidneys compared with normal controls. Gmfb expression was negatively associated with pathologic stage, T and M stages, and histologic grade. Univariate and multivariate analyses showed that elevated Gmfb expression was an independent factor for a favorable prognosis. Furthermore, the nomogram verified that Gmfb is a low-risk factor for KIRC. Knockdown of Gmfb in Caki-2 cells increased viability and decreased p21 and p27 levels. Overexpression of Gmfb inhibited Caki-2 cell proliferation, migration, and invasion and decreased mitochondrial membrane potential. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses considering Gmfb co-expressed differentially expressed genes (DEGs) showed that collecting duct acid secretion and mineral absorption ranked were the most important upregulated and downregulated DEGs, respectively. The upregulated hub genes for DEGs were mainly involved in nucleosome assembly, nucleosome organization, and chromatin assembly, and the downregulated hub genes were primarily associated with keratinization. The ratio of tumor-infiltrating immune cells in KIRC tissues was evaluated using CIBERSORTx. The results showed that the Gmfb expression was significantly positively correlated with macrophage M2 cells and mast resting cell infiltration levels and negatively correlated with T follicular helper, T regulatory, and B plasma cell infiltration levels. The former cell types were associated with a beneficial outcome, while the latter had a worse outcome in patients with KIRC. In summary, this study identified GMFB as a novel independent biomarker and therapeutic target for KIRC, and it provides a helpful and distinct individualized treatment strategy for KIRC with a combination of molecular targets and tumor microenvironment.

Melatonin Maintains Inner Blood-Retinal Barrier by Regulating Microglia via Inhibition of PI3K/Akt/Stat3/NF-κB Signaling Pathways in Experimental Diabetic Retinopathy.

Microglial activation and melatonin protection have been reported in diabetic retinopathy (DR). Whether melatonin could regulate microglia to protect the inner blood-retinal barrier (iBRB) remains unknown. In this study, the role of microglia in iBRB breakdown and the mechanisms of melatonin's regulation on microglia were explored. In diabetic rat retinas, activated microglia proliferated and migrated from the inner retina to the outer retina, accompanied by the obvious morphological changes. Meanwhile, significant leakage of albumin was evidenced at the site of close interaction between activated microglia and the damaged pericytes and endothelial cells. In vitro, inflammation-related cytokines, such as tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), interleukin (IL)-1ß, and arginase-1 (Arg-1), were increased significantly in CoCl2-treated BV2 cells. The supernatant derived from CoCl2-treated BV2 cells significantly decreased the cell viability and disrupted the junctional proteins in both pericytes and endothelial cells, resulting in severe leakage. Melatonin suppressed the microglial overactivation, i.e., decreasing the cell number and promoting its anti-inflammatory properties in diabetic rat retinas. Moreover, the leakage of iBRB was alleviated and the pericyte coverage was restored after melatonin treatment. In vitro, when treated with melatonin in CoCl2-treated BV2 cells, the inflammatory factors were decreased, while the anti-inflammatory factors were increased, further reducing the pericyte loss and increasing the tight junctions. Melatonin deactivated microglia via inhibition of PI3K/Akt/Stat3/NF-κB signaling pathways, thus maintaining the integrity of iBRB. The present data support a causal role for activated microglia in iBRB breakdown and highlight the therapeutic potential of melatonin in the treatment of DR by regulating microglia.

Glia Maturation Factor Beta as a Novel Biomarker and Therapeutic Target for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common types of cancer. The novel sensitive biomarkers and therapeutic targets are urgently needed for the early diagnosis of HCC and improvement of clinical outcomes. Glia maturation factor-ß (GMFB) is a growth and differentiation factor for both glia and neurons and has been found to be tightly involved in inflammation and neurodegeneration conditions. In our study, the expression level of GMFB was significantly up-regulated in patients with HCC and positively co-expression with tumor node metastases (TNM) stage and histopathological grade of HCC. The high expression level of GMFB was remarkably associated with poor overall survival, which mainly occurred in males rather than females. Multivariate analysis revealed GMFB to be an independent prognostic factor for overall survival in patients with HCC. Results of Gene Ontology (GO) and KEGG pathways analysis showed that down-regulation of pathways related to protein translation and mitochondria function were enriched. Protein-protein interaction analysis revealed the central role of mitochondria protein in HCC. The downregulation of genes involved in glycolysis and gluconeogenesis was observed among the co-expression genes of GMFB. Knockdown of GMFB in Hep3B significantly inhibited proliferation, migration, and invasion of Hep3B cells, and also downregulated the expression levels of some of metal matrix proteinase (MMP), increased mtDNA copy number and loss of mitochondrial transmembrane potential. GMFB influences the malignancy rate of HCC possibly through regulation of the expression of MMPs, mtDNA function and glycolysis. We proposed that GMFB was a promising HCC diagnostic and prognostic biomarker and therapeutic target in HCC.

Silencing Nogo-B improves the integrity of blood-retinal barrier in diabetic retinopathy via regulating Src, PI3K/Akt and ERK pathways.

OBJECTIVE: To examine the mechanisms of Nogo-B (RTN4B) in the protection of blood-retinal barrier in experimental diabetic retinopathy. METHODS: The level of Nogo-B in vitreous and plasma samples was detected with ELISA. Diabetes was induced in Sprague-Dawley rats with intraperitoneal injection of streptozotocin. The rats were injected intravitreally with adeno-associated virus (AAV) for knockdown the expression of Nogo-B in retina or/and as AAV negative control. The permeability of blood-retinal barrier was detected with Rhodamine-B-dextran leakage assay. The expressions of Nogo-B, junctional proteins, inflammatory factors and signaling pathways were examined with Western blot and quantitative real-time PCR. RESULTS: Nogo-B expression was significantly upregulated in clinical samples and experimental diabetic rat models. Under normal condition, Nogo-B knockdown resulted in the increased permeability of retinal blood vessels. In diabetic rat retinas, the vascular leakage was increased significantly, which was partially decreased by Nogo-B knockdown through increasing p/t-Src (Tyr529) and p/t-Akt (Ser473), and decreasing p/t-ERK1/2. CONCLUSION: Nogo-B was increased in diabetic retinopathy and silencing Nogo-B is a promising therapy for diabetic retinopathy.

BMSC-derived extracellular vesicles intervened the pathogenic changes of scleroderma in mice through miRNAs.

BACKGROUND: Systemic sclerosis (SSc) is a disease that features severe fibrosis of the skin and lacks effective therapy. Bone marrow mesenchymal stem cell (BMSC)-derived extracellular vesicles (EVs) are potential stem cell-based tools for the treatment of SSc. METHODS: BMSCs were isolated from the bone marrow of mice and identified with surface markers according to multilineage differentiation. EVs were isolated from the BMSC culture medium by ultracentrifugation and identified with a Nanosight NS300 particle size analyzer, transmission electron microscopy (TEM), and western blot. The microRNAs (miRNAs) of BMSC-derived EVs (BMSC-EVs) were studied via miRNA sequencing (miRNA-seq) and bioinformatic analysis. An SSc mouse model was established via subcutaneous bleomycin (BLM) injection, and the mice were treated with BMSCs or BMSC-derived EVs. Skin tissues were dissociated and analyzed with H&E staining, RNA sequencing (RNA-seq), western blot, and immunohistochemical staining. RESULTS: Evident pathological changes, like fibrosis and inflammation, were induced in the skin of BLM-treated mice. BMSCs and BMSC-EVs effectively intervened such pathological manifestations and disease processes in a very similar way. The effects of the BMSC-EVs were found to be caused by the miRNAs they carried, which were proven to be involved in regulating the proliferation and differentiation of multiple cell types and in multiple EV-related biological processes. Furthermore, TGF-ß1-positive cells and α-SMA-positive myofibroblasts were significantly increased in the scleroderma skin of BLM-treated mice but evidently reduced in the scleroderma skin of the EV-treated SSc group. In addition, the numbers of mast cells and infiltrating macrophages and lymphocytes were evidently increased in the skin of BLM-treated mice but significantly reduced by EV treatment. In line with these observations, there were significantly higher mRNA levels of the inflammatory cytokines Il6, Il10, and Tnf-α in SSc mice than in control mice, but the levels decreased following EV treatment. Through bioinformatics analysis, the TGFß and WNT signaling pathways were revealed to be closely involved in the pathogenic changes seen in mouse SSc, and these pathways could be therapeutic targets for treating the disease. CONCLUSIONS: BMSC-derived EVs could be developed as a potential therapy for treating skin dysfunction in SSc, especially considering that they show similar efficacy to BMSCs but have fewer developmental regulatory requirements than cell therapy. The effects of EVs are generated by the miRNAs they carry, which alleviate SSc pathogenic changes by regulating the WNT and TGFß signaling pathways.

Inhibition of PARP activity improves therapeutic effect of ARPE-19 transplantation in RCS rats through decreasing photoreceptor death.

Photoreceptor (PR) dysfunction or death is the key pathological change in retinal degeneration (RD). The death of PRs might be due to a primary change in PRs themselves or secondary to the dysfunction of the retinal pigment epithelium (RPE). Poly(ADP-ribose) polymerase (PARP) was reported to be involved in primary PR death, but whether it plays a role in PR death secondary to RPE dysfunction has not been determined. To clarify this question and develop a new therapeutic approach, we studied the changes in PAR/PARP in the RCS rat, a RD model, and tested the effect of PARP intervention when given alone or in combination with RPE cell transplantation. The results showed that poly(ADP-ribosyl)ation of proteins was increased in PRs undergoing secondary death in RCS rats, and this result was confirmed by the observation of similar changes in sodium iodate (SI)-induced secondary RD in SD rats. The increase in PAR/PARP was highly associated with increased apoptotic PRs and decreased visual function, as represented by lowered b-wave amplitudes on electroretinogram (ERG). Then, as we expected, when the RCS rats were treated with subretinal injection of the PARP inhibitor PJ34, the RD process was delayed. Furthermore, when PJ34 was given simultaneously with subretinal ARPE-19 cell transplantation, the therapeutic effects were significantly improved and lasted longer than those of ARPE-19 or PJ34 treatment alone. These results provide a potential new approach for treating RD.

Erythropoietin protects the inner blood-retinal barrier by inhibiting microglia phagocytosis via Src/Akt/cofilin signalling in experimental diabetic retinopathy.

AIMS/HYPOTHESIS: Microglial activation in diabetic retinopathy and the protective effect of erythropoietin (EPO) have been extensively studied. However, the regulation of microglia in the retina and its relationship to inner blood-retinal barrier (iBRB) maintenance have not been fully characterised. In this study, we investigated the role of microglia in iBRB breakdown in diabetic retinopathy and the protective effects of EPO in this context. METHODS: Male Sprague Dawley rats were injected intraperitoneally with streptozotocin (STZ) to establish the experimental model of diabetes. At 2 h after STZ injection, the right and left eyes were injected intravitreally with EPO (16 mU/eye, 2 µl) and an equivalent volume of normal saline (NaCl 154 mmol/l), respectively. The rats were killed at 2 or 8 weeks after diabetes onset. Microglia activation was detected by ionised calcium binding adaptor molecule (IBA)-1 immunolabelling. Leakage of the iBRB was evaluated by albumin staining and FITC-dextran permeability assay. BV2 cells and primary rat microglia under hypoxic conditions were used to model microglial activation in diabetic retinopathy. Phagocytosis was examined by confocal microscopy in flat-mounted retina preparations and in microglia and endothelial cell cocultures. Protein levels of IBA-1, CD11b, complement component 1r (C1r), and Src/Akt/cofilin signalling pathway components were assessed by western blotting. RESULTS: In diabetic rat retinas, phagocytosis of endothelial cells by activated microglia was observed at 8 weeks, resulting in an increased number of acellular capillaries (increased by 426.5%) and albumin leakage. Under hypoxic conditions, activated microglia transmigrated to the opposite membrane of the transwell, where they disrupted the endothelial cell monolayer by engulfing endothelial cells. The activation and phagocytic activity of microglia was blocked by intravitreal injection of EPO. In vitro, IBA-1, CD11b and C1r protein levels were increased by 50.9%, 170.0% and 135.5%, respectively, by hypoxia, whereas the phosphorylated proteins of Src/Akt/cofilin signalling pathway components were decreased by 74.2%, 47.8% and 39.7%, respectively, compared with the control; EPO treatment abrogated these changes. CONCLUSIONS/INTERPRETATION: In experimental diabetic retinopathy, activated microglia penetrate the basement membrane of the iBRB and engulf endothelial cells, leading to iBRB breakdown. EPO exerts a protective effect that preserves iBRB integrity via activation of Src/Akt/cofilin signalling in microglia, as demonstrated in vitro. These data support a causal role for activated microglia in iBRB breakdown and highlight the therapeutic potential of EPO for the treatment of diabetic retinopathy. Graphical abstract.

Erythropoietin maintains VE-cadherin expression and barrier function in experimental diabetic retinopathy via inhibiting VEGF/VEGFR2/Src signaling pathway.

AIMS: To explore the mechanisms of erythropoietin (EPO)'s protection on inner blood-retinal barrier (iBRB) in experimental diabetic retinopathy. MATERIAL AND METHODS: Male SD rats were rendered diabetic with streptozotocin, followed by intravitreal injection of EPO. The permeability of iBRB was examined with fluorescein isothiocyanate (FITC)-dextran. Human retinal microvascular endothelial cells (HRMECs) and human umbilical vein endothelial cells (HUVECs) were treated with glyoxal and studied for cell viability and barrier function. The expressions of vascular endothelial (VE)-cadherin, Src kinase, vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR2) were analyzed with Western blot, ELISA, qPCR, or immunofluorescence. KEY FINDINGS: VE-cadherin in rat retinas was down-regulated with diabetes progression. EPO treatment could increase VE-cadherin expression at week 8 and week 16. The expressions of p-Src and p-VE-cadherin were increased at week 2, while decreased at week 8 of diabetes; which were prevented by EPO. The leakage of FITC-dextran in 8-week diabetic rat retinas was ameliorated by EPO. In vitro results showed the expressions of VEGF, p-Src and p-VE-cadherin were increased significantly, accompanied with the decreased barrier function, which were prevented by EPO. Ranibizumab and CGP77675 also inhibited the glyoxal-induced phosphorylation of Src and VE-cadherin. Cellular fractionation showed EPO mitigated the VE-cadherin internalization in glyoxal-treated cells. SIGNIFICANCE: EPO maintained the expression of VE-cadherin in experimental diabetic retinopathy by inhibiting its phosphorylation and internalization through VEGF/VEGFR2/Src pathway, thus improved the integrity of iBRB.

Cytokines Induce Monkey Neural Stem Cell Differentiation through Notch Signaling.

The mammalian central nervous system (CNS) has a limited ability to renew the damaged cells after a brain or spinal cord injury whether it is nonhuman primates like monkeys or humans. Transplantation of neural stem cells (NSCs) is a potential therapy for CNS injuries due to their pluripotency and differentiation abilities. Cytokines play an important role in CNS development and repair of CNS injuries. However, the detailed cytokine signaling response in monkey neural stem cells is rarely studied. In our previous research, we isolated NSCs from the adult monkey brain and found the effects of cytokines on monkey NSCs. Now, we further analyzed the regulation mechanisms of cytokines to the proliferation of monkey NSCs such as bone morphogenic protein 4 (BMP4), BMP4/leukaemia inhibitory factor (LIF), or retinoic acid (RA)/Forskolin. The data showed that BMP4 inhibited cell proliferation to arrest, but it did not affect the stemness of NSCs. BMP4/LIF promoted the astrocyte-like differentiation of monkey NSCs, and RA/forskolin induced the neuronal differentiation of monkey NSCs. BMP4/LIF and RA/forskolin induced monkey NSC differentiation by regulating Notch signaling. These results provide some theoretical evidence for NSC therapy to brain or spinal cord injury in regenerative medicine.

Transplantation Site Affects the Outcomes of Adipose-Derived Stem Cell-Based Therapy for Retinal Degeneration.

Adipose-derived stem cells (ASCs) have shown a strong protective effect on retinal degenerative diseases (RDD) after being transplanted into the subretinal space in an animal model. Recently, several clinical trials have been conducted to treat RDD with intravitreal transplantation of stem cells, including ASCs. However, the outcomes of the clinical trials were not satisfactory. To investigate if the transplantation site alters the outcome of stem cell-based therapy for RDD, we isolated rat ASCs (rASCs) and labeled them with green fluorescent protein. Autologous rASCs were grafted into the vitreous chamber or subretinal space in a rat RDD model induced by sodium iodate (SI). The electric response was recorded by ERG. The anatomic structure of the retina was observed in cryosections of rat eyes at posttransplantation weeks 1, 2, and 4. Neural retina apoptosis and epiretinal membrane- (ERM-) like structure formation were investigated by immunostaining. The intravitreal transplantation of rASCs resulted in an extinguished electric response, although the rosette formation and apoptosis of neural retina were reduced. However, the rASCs that grafted in the subretinal space protected the retina from the damage caused by SI, including a partial recovering of the electric response and a reduction in rosette formation. Intravitreally grafted rASCs formed a membrane, resulting in retina folding at the injection site. Müller cells, retinal pigment epithelial cells, and microglial cells migrated from the retina to the rASC-formed membrane and subsequently formed an ERM-like structure. Furthermore, vitreous fluid promoted rASC migration, and rASC-conditioned medium enhanced Müller cell migration as indicated by in vitro studies. These data suggested that the vitreous chamber is not a good transplantation site for ASC-based therapy for RDD and that a deliberate decision should be made before transplantation of stem cells into the vitreous chamber to treat RDD in clinical trials.

Protein Kinase A Inhibitor H89 Attenuates Experimental Proliferative Vitreoretinopathy.

Purpose: This study aimed to explore the role of the protein kinase A (PKA) pathway in proliferative vitreoretinopathy (PVR) and the effect of the PKA inhibitor H89 on experimental PVR. Methods: Epiretinal membranes (ERMs) were acquired from PVR patients and analyzed by frozen-section immunofluorescence. An in vivo model was developed by intravitreal injecting rat eyes with ARPE-19 cells and platelet-rich plasma, and changes in eye structures and vision function were observed. An in vitro epithelial-mesenchymal transition (EMT) cell model was established by stimulating ARPE-19 cells with transforming growth factor (TGF)-ß. Alterations in EMT-related genes and cell function were detected. Mechanistically, PKA activation and activity were explored to assess the relationship between TGF-ß1 stimulation and the PKA pathway. The effect of H89 on the TGF-ß-Smad2/3 pathway was detected. RNA sequencing was used to analyze gene expression profile changes after H89 treatment. Results: PKA was activated in human PVR membranes. In vivo, H89 treatment protected against structural changes in the retina and prevented decreases in electroretinogram b-wave amplitudes. In vitro, H89 treatment inhibited EMT-related gene alterations and partially reversed the functions of the cells. TGF-ß-induced PKA activation was blocked by H89 pretreatment. H89 did not affect the phosphorylation or nuclear translocation of regulatory Smad2/3 but increased the expression of inhibitory Smad6. Conclusions: PKA pathway activation is involved in PVR pathogenesis, and the PKA inhibitor H89 can effectively inhibit PVR, both in vivo and in vitro. Furthermore, the protective effect of H89 is related to an increase in inhibitory Smad6.

The combination of bFGF and CHIR99021 maintains stable self-renewal of mouse adult retinal progenitor cells.

BACKGROUND: Millions of people are affected with retinal diseases that eventually cause blindness, and retinal progenitor cell (RPC) transplantation is a promising therapeutic avenue. However, RPC expansion and the underlying regulation mechanisms remain elusive. METHODS: Adult mouse neural RPCs (mNRPCs) were isolated and amplified with the combination of basic fibroblast growth factor (bFGF) and glycogen synthase kinase 3 (GSK3) inhibitor CHIR99021. The progenitor characteristics were evaluated with RT-PCR, immunocytochemistry (ICC), western blot, flow cytometry, and transcriptome analysis prior to transplantation. By treating cells with or without bFGF and CHIR99021 at different time points, the mechanism for mNRPCs' self-renewal was investigated by transcriptome analysis and western blot assay. RESULTS: mNRPCs were self-renewing in the presence of bFGF and CHIR99021 and showed prominent RPC characteristics. bFGF was essential in promoting cell cycle by facilitating G1/S and G2/M transitions. bFGF combined with CHIR99021 activated the non-canonical Wnt5A/Ca2+ pathway and form a calcium homeostasis. In addition, the self-renewing mNRPCs could differentiate into rod photoreceptor-like cells and retinal pigment epithelium (RPE)-like cells by in vitro induction. When green fluorescent protein (GFP)-labeled cells were transplanted into the subretinal space (SRS) of Pde6b (rd1) mice (also known as RD1 mice, or rodless mice), the cells survived for more than 12 weeks and migrated into the retina. Parts of the recipient retina showed positive expression of photoreceptor marker rhodopsin. Transplanted cells can migrate into the retina, mainly into the inner cell layer (INL) and ganglion cell layer (GCL). Some cells can differentiate into astrocytes and amacrine cells. Cultured mNRPCs did not form tumors after transplanted into NOD/SCID mice for 6 months. CONCLUSIONS: Present study developed an approach to maintain long-term self-renewal of RPCs from adult retinal tissues and revealed that activation of the non-canonical Wnt5A/Ca2+ pathway may participate in regulating RPC self-renewal in vitro. This study presents a very promising platform to expand RPCs for future therapeutic application.