Furong Gao

Subretinal Delivery of AAV2-Mediated Human Erythropoietin Gene Is Protective and Safe in Experimental Diabetic Retinopathy

PURPOSE We studied and developed a gene-based intraocular erythropoietin (EPO) therapy for diabetic retinopathy (DR), by which the applicability of neuroprotective therapy with favorable safety profile is attempted. METHODS Hematocrit (Hct) was measured in C57BL/6 mice after intramuscular injection of AAV2-CMV-hEPO virus. Diabetes was induced by intraperitoneal injection of streptozotocin in Sprague-Dawley (SD) rats. Subretinal or intravitreal injection was performed in SD rats and Dark Agouti (DA) rats. The human EPO (hEPO) concentration was measured with ELISA. Blood-retinal barrier (BRB) breakdown was measured with Evans blue permeation. Retinal function was evaluated with electroretinography (ERG). Retinal cell apoptosis was detected with TUNEL. Retinal thickness and cell counts were examined by light microscopy. Retinal vascular changes were evaluated with fundus fluorescein angiography (FFA) and confocal microscopy. RESULTS The serum hEPO was elevated 2 weeks after AAV2-CMV-hEPO virus injection, and Hct began to increase after 4 weeks. After subretinal injection, hEPO expressions in aqueous humor, vitreous, and retina followed a dose- and time-dependent manner. In the AAV2-CMV-hEPO-treated diabetic group, BRB was maintained, and retinal cell apoptosis was significantly reduced. The ERG results showed that the retinal function remained unchanged for at least one year after subretinal injection of AAV2-CMV-hEPO virus. Long-term expression of hEPO following subretinal injection of AAV2-CMV-hEPO virus did not induce neovascularization in retina and choroid. CONCLUSIONS The AAV2-CMV-hEPO gene therapy is safe, and it exerts long-term protective effects on diabetic retinas. Thus, the gene therapy by using AAV2-CMV-hEPO for DR is feasible.

Human Adipose-Derived Stem Cells Delay Retinal Degeneration in Royal College of Surgeons Rats Through Anti-Apoptotic and VEGF-Mediated Neuroprotective Effects

UNLABELLED Stem cell therapy is a promising therapeutic approach for retinal degeneration (RD). Our study investigated the effects of human adipose derived stem cell (hADSCs) on Royal College of Surgeons (RCS) rats. METHODS Green fluorescent protein (GFP)-labeled hADSCs were transplanted subretinally into RCS rats at postnatal (PN) 21 days to explore potential therapeutic effects, while adeno-associated viral vector (AAV2)-vascular endothelial growth factor (VEGF) and siVEGF-hADSCs were used to aid the mechanistic dissections. Visual function was evaluated by Electroretinogram (ERG) recording. Potential transdifferentiations were examined by Immunofluorescence (IF) and gene expressions were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Apoptotic retinal cells were detected by Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) assay and the cytokines secreted by hADSCs were measured by Enzyme-linked Immunosorbent Assay (ELISA). RESULTS The visual function of RCS rats began to decrease one week after their eyes opened at PN week 3 and almost lost in PN 5 weeks, accompanied by the loss of retinal outer nuclear layer (ONL). Subretinal transplantation of hADSCs significantly improved the visual function 2 weeks after the transplantation and such therapeutic effect persisted up to 8 weeks after the treatment (PN 11 weeks), with 3-4 rows of photoreceptors remained in the ONL and reduced apoptosis. Consistent with these phenotypic changes, the gene expression of rod photoreceptor markers Rhodopsin (Rho), Crx and Opsin (Opn1) in RCS rats showed obvious decreasing trends over time after PN 3 weeks, but were elevated with hADSC treatment. hADSC transplantation also repressed the expressions of Bax, Bak and Caspase 3, but not the expression of anti-apoptotic genes, including Bcl-2 and Bcl-XL. Finally, substantial VEGF, hepatocyte growth factor (HGF) and pigment epithelium-derived factor (PEDF) secretions from hADSCs were detected, while endogenous Vegf expression level decreased over time in RCS rats. The treatment of AAV2-VEGF showed comparable therapeutic effects as hADSCs but siRNA knockdown of VEGF in hADSCs essentially abolished the therapeutic effects. CONCLUSIONS Subretinal transplantation of hADSCs in RCS rats effectively delayed the retinal degeneration, enhanced the retinal cell survival and improved the visual function. Mechanistically this was mainly due to hADSC dependent anti-apoptotic and neuroprotective effects through its secretion of growth and neurotrophic factors including VEGF. Clinical application of hADSCs merits further investigation.

Subpopulations of bone marrow mesenchymal stem cells exhibit differential effects in delaying retinal degeneration.

Bone marrow mesenchymal stem cells (BMSCs) have a therapeutic role in retinal degeneration (RD). However, heterogeneity of BMSCs may be associated with differential therapeutic effects in RD. In order to confirm this hypothesis, two subsets of rat BMSCs, termed rBMSC1 and rBMSC2, were obtained, characterized and functionally evaluated in the treatment of RD of Royal College of Surgeons (RCS) rats. Both subpopulations expressed mesenchymal stem cells (MSC) markers CD29 and CD90, but were negative for hemacyte antigen CD11b and CD45 expression. In comparison with rBMSC2, rBMSC1 showed higher rate of proliferation, stronger colony formation, and increased adipogenic potential, whereas rBMSC2 exhibited higher osteogenic potential. Microarray analysis showed differential gene expression patterns between rBMSC1 and rBMSC2, including functions related to proliferation, differentiation, immunoregulation, stem cell maintenance and division, survival and antiapoptosis. After subretinal transplantation in RCS rats, rBMSC1 showed stronger rescue effect than rBMSC2, including increased b-wave amplitude, restored retinal nuclear layer thickness, and decreased number of apoptotic photoreceptors, whereas the rescue function of rBMSC2 was essentially not better than the control. Histological analysis also demonstrated that rBMSC1 possessed a higher survival rate than rBMSC2 in subretinal space. In addition, treatment of basic fibroblast growth factor, an accompanying event in subretinal injection, triggered more robust increase in secretion of growth factors by rBMSC1 as compared to rBMSC2. Taken together, these results have suggested that the different therapeutic functions of BMSC subpopulations are attributed to their distinct survival capabilities and paracrine functions. The underlying mechanisms responsible for the different functions of BMSC subpopulation may lead to a new strategy for the treatment of RD.

The usability of WeChat as a mobile and interactive medium in student‐centered medical teaching

Biochemistry and cellular biology courses for medical students at Tongji University include the assessment that provides students with feedback to enhance their learning, which is a type of formative assessment. However, frequent instant feedback and guidance for students is often absent or inconsistently included in the teaching process. WeChat, the most popular Chinese social media, was introduced in biochemistry and cellular biology course. A WeChat official account (OA) was set up as an instant interactive platform. Over a period of two semesters, OA sent 73 push notifications. The components included course notices, preclass thought questions, after‐class study materials, answer questions and feedback, simulation exercises, teacher–student interaction, and research progress relevant to the course. WeChat OA served as an active‐learning teaching tool, provided more frequent feedback and guidance to students, and facilitated better student‐centered communication in the teaching process. Using the WeChat OA in medical teaching emphasized interactive, interoperable, effective, engaging, adaptable, and more participatory teaching styles. As a new platform, WeChat OA was free, Internet‐reliant, and easily managed. Using this new medium as a communication tool accelerated further advancement of instant feedback and improvement in teaching activities. Notifications and interactive feedback via the mobile social medium WeChat OA anytime and anywhere facilitated a student‐centered teaching mode. Use of WeChat OA significantly increased the proportion of students interactively participating and resulted in a high degree of student satisfaction.

A cell culture condition that induces the mesenchymal-epithelial transition of dedifferentiated porcine retinal pigment epithelial cells

&NA; The pathological change of retinal pigment epithelial (RPE) cells is one of the main reasons for the development of age‐related macular degeneration (AMD). Thus, cultured RPE cells are a proper cell model for studying the etiology of AMD in vitro. However, such cultured RPE cells easily undergo epithelial‐mesenchymal transition (EMT) that results in changes of cellular morphology and functions of the cells. To restore and maintain the mesenchymal‐epithelial transition (MET) of the cultured RPE cells, we cultivated dedifferentiated porcine RPE (pRPE) cells and compared their behaviors in four conditions: 1) in cell culture dishes with DMEM/F12 containing FBS (CC dish‐FBS), 2) in petri dishes with DMEM/F12 containing FBS (Petri dish‐FBS), 3) in cell culture dishes with DMEM/F12 containing N2 and B27 supplements (CC dish‐N2B27), and 4) in petri dishes with DMEM/F12 containing N2 and B27 (Petri dish‐N2B27). In addition to observing the cell morphology and behavior, RPE specific markers, as well as EMT‐related genes and proteins, were examined by immunostaining, quantitative real‐time PCR and Western blotting. The results showed that dedifferentiated pRPE cells maintained EMT in CC dish‐FBS, Petri dish‐FBS and CC dish‐N2B27 groups, whereas MET was induced when the dedifferentiated pRPE cells were cultured in Petri dish‐N2B27. Such induced pRPE cells showed polygonal morphology with increased expression of RPE‐specific markers and decreased EMT‐associated markers. Similar results were observed in induced pluripotent stem cell‐derived RPE cells. Furthermore, during the re‐differentiation of those dedifferentiated pRPE cells, Petri dish‐N2B27 reduced the activity of RhoA and induced F‐actin rearrangement, which promoted the nuclear exclusion of transcriptional co‐activator with PDZ‐binding motif (TAZ) and TAZ target molecule zinc finger E‐box binding protein (ZEB1), both of which are EMT inducing factors. This study provides a simple and reliable method to reverse dedifferentiated phenotype of pRPE cells into epithelialized phenotype, which is more appropriate for studying AMD in vitro, and suggests that MET of other cell types might be induced by a similar approach.

Pseudomonas aeruginosa infection alters the macrophage phenotype switching process during wound healing in diabetic mice: P. aeruginosa regulates macrophage phenotype

Macrophages play critical roles in wound healing process. They switch from “classically activated” (M1) phenotype in the early inflammatory phase to “alternatively activated” (M2) phenotype in the later healing phase. However, the dynamic process of macrophage phenotype switching in diabetic wounds burdened with bacteria is unclear. In this report, Pseudomonas aeruginosa, frequently detected in diabetic foot ulcers, was inoculated into cutaneous wounds of db/db diabetic mice to mimic bacterium‐infected diabetic wound healing. We observed that P. aeruginosa infection impaired diabetic wound healing and quickly promoted the expression of pro‐inflammatory genes (M1 macrophage markers) tumor necrosis factor‐α (tnf‐α), interleukin‐1β (il‐1β) and il‐6 in wounds. The expression of markers of M2 macrophages, including il‐10, arginase‐1, and ym1 were also upregulated. In addition, similar gene expression patterns were observed in macrophages isolated directly from wounds. Immunostaining showed that P. aeruginosa infection increased both the ratios of M1 and M2 macrophages in wounds compared with that in control groups, which was further confirmed by in vitro culturing macrophages with P. aeruginosa and skin fibroblast conditioned medium. However, the ratios of the expression levels of pro‐inflammatory genes to anti‐inflammatory gene il‐10 was increased markedly in P. aeruginosa infected wounds and macrophages compared with that in control groups, and P. aeruginosa prolonged the presence of M1 macrophages in the wounds. These data demonstrated that P. aeruginosa in diabetic wounds activates a mixed M1/M2 macrophage phenotype with an excessive activation of M1 phenotype or relatively inadequate activation of M2 phenotype.

miR-365 promotes diabetic retinopathy through inhibiting Timp3 and increasing oxidative stress

ABSTRACT miRs play critical roles in oxidative stress‐related retinopathy pathogenesis. miR‐365 was identified in a previously constructed library from glyoxal‐treated rat Müller cell. This report explores epigenetic alterations in Müller cells under oxidative stress to develop a novel therapeutic strategy. To examine the miR‐365 expression pattern, in situ hybridization and quantitative RT‐PCR were performed. Bioinformatical analysis and dual luciferase report assay were applied to identify and confirm target genes. Streptozotocin (STZ)‐treated rats were used as the diabetic retinopathy (DR) model. Lentivirus‐mediated anti‐miR‐365 was delivered subretinally and intravitreally into the rats’ eyes. The functional and structural changes were evaluated by electroretinogram (ERG), histologically, and through examination of expression levels of metallopeptidase inhibitor 3 (Timp3), glial fibrillary acidic protein (Gfap), recoverin (Rcvrn) and vascular endothelia growth factor A (Vegfa). Oxidative stress factors and pro‐inflammatory cytokines were analyzed. miR‐365 expression was confirmed in the glyoxal‐treated rat Müller cell line (glyoxal‐treated rMC‐1). In the retina, miR‐365 mainly localized in the inner nuclear layer (INL). The increased miR‐365 participated in Müller cell gliosis through oxidative stress aggravation, as observed in glyoxal‐treated rMC‐1 and DR rats before 6 weeks. Timp3 was a target and negatively regulated by miR‐365. When miR‐365 was inhibited, Timp3 expression was upregulated, Müller cell gliosis was alleviated, and retinal oxidative stress was attenuated. Visual function was also partially rescued as detected by ERG. miR‐365 was found to be highly expressed in the retina and the abnormality of miR‐365/Timp3 pathway is closely related to the pathology, like Müller gliosis, and the visual injury in DR. The mechanism might be through oxidative stress, and miR‐365/Timp3 could be a potential therapeutic target for treating DR. HIGHLIGHTSmiR‐365 in rat retina is mainly expressed in INL, photoreceptor layer, and RPE layer.Timp3 is the direct target of and negatively regulated by miR‐365.The increased miR‐365 in Müller cells is involved in DR through miR‐365/Timp3 pathway and oxidative stress mechanism.

Human Umbilical Cord Mesenchymal Stem Cells: Subpopulations and Their Difference in Cell Biology and Effects on Retinal Degeneration in RCS Rats

BACKGROUND Human umbilical cord mesenchymal stem cells (hUC-MSCs) are potential candidates for treating retinal degeneration (RD). OBJECTIVE To further study the biology and therapeutic effects of the hUC-MSCs on retinal degeneration. METHODS Two hUC-MSC subpopulations, termed hUC-MSC1 and hUC-MSC2, were isolated by single-cell cloning method and their therapeutic functions were compared in RCS rat, a RD model. RESULTS Although both subsets satisfied the basic requirements for hUC-MSCs, they were significantly different in morphology, proliferation rate, differentiation capacity, phenotype and gene expression. Furthermore, only the smaller, fibroblast-like, faster growing subset hUC-MSC1 displayed stronger colony forming potential as well as adipogenic and osteogenic differentiation capacities. When the two subsets were respectively transplanted into the subretinal spaces of RCS rats, both subsets survived, but only hUC-MSC1 expressed RPE cell markers Bestrophin and RPE65. More importantly, hUC-MSC1 showed stronger rescue effect on the retinal function as indicated by the higher b-wave amplitude on ERG examination, thicker retinal nuclear layer, and decreased apoptotic photoreceptors. When both subsets were treated with interleukin-6, mimicking the inflammatory environment when the cells were transplanted into the eyes with degenerated retina, hUC-MSC1 expressed much higher levels of trophic factors in comparison with hUC-MSC2. CONCLUSION The data here, in addition to prove the heterogeneity of hUC-MSCs, confirmed that the stronger therapeutic effects of hUC-MSC1 were attributed to its stronger anti-apoptotic effect, paracrine of trophic factors and potential RPE cell differentiation capacity. Thus, the subset hUC-MSC1, not the other subset or the ungrouped hUC-MSCs should be used for effective treatment of RD.