Proteomic study of vitreous in proliferative diabetic retinopathy patients after treatment with aflibercept: a quantitative analysis based on 4D label-free technique.

AIM: To identify different metabolites, proteins and related pathways to elucidate the causes of proliferative diabetic retinopathy (PDR) and resistance to anti-vascular endothelial growth factor (VEGF) drugs, and to provide biomarkers for the diagnosis and treatment of PDR. METHODS: Vitreous specimens from patients with diabetic retinopathy were collected and analyzed by Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analyses based on 4D label-free technology. Statistically differentially expressed proteins (DEPs), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway representation and protein interactions were analyzed. RESULTS: A total of 12 samples were analyzed. The proteomics results showed that a total of 58 proteins were identified as DEPs, of which 47 proteins were up-regulated and 11 proteins were down-regulated. We found that C1q and tumor necrosis factor related protein 5 (C1QTNF5), Clusterin (CLU), tissue inhibitor of metal protease 1 (TIMP1) and signal regulatory protein alpha (SIRPα) can all be specifically regulated after aflibercept treatment. GO functional analysis showed that some DEPs are related to changes in inflammatory regulatory pathways caused by PDR. In addition, protein-protein interaction (PPI) network evaluation revealed that TIMP1 plays a central role in neural regulation. In addition, CD47/SIRPα may become a key target to resolve anti-VEGF drug resistance in PDR. CONCLUSION: Proteomic analysis is an approach of choice to explore the molecular mechanisms of PDR. Our data show that multiple proteins are differentially changed in PDR patients after intravitreal injection of aflibercept, among which C1QTNF5, CLU, TIMP1 and SIRPα may become targets for future treatment of PDR and resolution of anti-VEGF resistance.

Sequence Control in Asymmetric Terpolymerizations of meso-Epoxides, CO2 , and Phthalic Anhydride: Unprecedented Statistical Ester-Carbonate Distributions.

The statistical terpolymerization of epoxides, CO2 and cyclic anhydrides remains challenging, mainly because epoxide/CO2 and epoxide/anhydride copolymerizations typically proceed at considerably different rates. Herein, we report the syntheses of novel chiral terpolymers with unprecedented statistical distributions of carbonate and ester units (up to 50 % junction units) via the one-pot reaction of cyclohexene oxide, phthalic anhydride, and CO2 under mild conditions using enantiopure bimetallic aluminum-complex-based catalyst systems. Notably, all resulting terpolymers exhibited excellent enantioselectivities (≥96 % ee) that were independent of the carbonate-ester distribution. The statistical compositions of the carbonate and ester units in the resulting terpolymers were determined via 1 H and 13 C NMR spectroscopies. Furthermore, thermal properties were tuned by altering the ester content of the chiral terpolymer without influencing the enantioselective ring-opening step involving the meso-epoxide. This asymmetric terpolymerization methodology is also compatible with a variety of meso-epoxides to afford the corresponding terpolymers with 17 %-25 % junction units and excellent enantioselectivities (94 %-99 % ee). The present study is expected to provide new guidelines for preparing a broad range of biodegradable polymers with excellent enantioselectivities and adjustable properties.

Physical exercise attenuates age-related muscle atrophy and exhibits anti-ageing effects via the adiponectin receptor 1 signalling.

BACKGROUND: Although the adiponectin signalling exerts exercise-mimicking effects, whether this pathway contributes to the anti-ageing benefits of physical exercise has not been established yet. METHODS: Swim exercise training and wheel running were used to measure lifespan in the nematode Caenorhabditis elegans and skeletal muscle quality in mice, respectively. Muscle weight, muscle fibre cross-sectional area (CSA) and myonuclei number were used to evaluate muscle mass. RNA sequencing (RNA-Seq) analysis of skeletal muscle in exercised mice was used to study the underlying mechanisms. Western blot and immunofluorescence were performed to explore autophagy- and senescence-related markers. RESULTS: The C. elegans adiponectin receptor PAQR-1/AdipoR1, but not PAQR-2/AdipoR2, was activated (3.55-fold and 3.48-fold increases in p-AMPK on Days 1 and 6, respectively, P < 0.001), which was involved in lifespan extension in exercised worms. Exercise training increased skeletal muscle mass index (1.29-fold, P < 0.01), muscle weight (1.75-fold, P < 0.001), myonuclei number (1.33-fold, P < 0.05), muscle fibre CSA (1.39-fold, P < 0.05) and capillary abundance (2.19-fold, P < 0.001 for capillary density; 1.58-fold, P < 0.01 for capillary number) in aged mice. Physical exercise reduced protein (2.94-fold, P < 0.001) and mRNA levels (1.70-fold, P < 0.001) of p16INK4a , a marker for cellular senescence, in skeletal muscle of aged mice. These beneficial effects of exercise on skeletal muscle of mice were dependent on AdipoR1. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for differentially expressed genes in skeletal muscle between exercised mice with and without AdipoR1 knockdown by RNA-Seq analysis revealed that several KEGG pathways, such as 'AMPK signalling pathway' (P < 0.001), 'FOXO signalling pathway' (P < 0.001) and 'autophagy' (P < 0.001) were overrepresented. Knockdown of FoxO3a inhibited exercise-mediated beneficial effects on skeletal muscle quality of mice by inhibiting autophagy/mitophagy (3.81-fold reduction in LC3-II protein, P < 0.001; 1.53-fold reduction in BNIP3 protein, P < 0.05). Knockdown of daf-16, the FoxO homologue in C. elegans, reduced autophagy (2.77-fold and 2.06-fold reduction in GFP::LGG-1 puncta in seam cells and the intestine, respectively, P < 0.05) and blocked lifespan extension by exercise in worms. CONCLUSIONS: Our findings provide insights into how the AdipoR1 pathway has an impact on the anti-ageing benefits of exercise and implicate that activation of the AdipoR1 signalling may represent a potential therapeutic strategy for reducing age-related loss of skeletal muscle.

Mesenchymal stem cell-derived exosomes inhibit the VEGF-A expression in human retinal vascular endothelial cells induced by high glucose.

AIM: To determine the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells (hUCMSCs) on the expression of vascular endothelial growth factor A (VEGF-A) in human retinal vascular endothelial cells (HRECs). METHODS: Exosomes were isolated from hUCMSCs using cryogenic ultracentrifugation and characterized by transmission electron microscopy, Western blotting and nanoparticle tracking analysis. HRECs were randomly divided into a normal control group (group A), a high glucose model group (group B), a high glucose group with 25 µg/mL (group C), 50 µg/mL (group D), and 100 µg/mL exosomes (group E). Twenty-four hours after coculture, the cell proliferation rate was detected using flow cytometry, and the VEGF-A level was detected using immunofluorescence. After coculture 8, 16, and 24h, the expression levels of VEGF-A in each group were detected using PCR and Western blots. RESULTS: The characteristic morphology (membrane structured vesicles) and size (diameter between 50 and 200 nm) were observed under transmission electron microscopy. The average diameter of 122.7 nm was discovered by nanoparticle tracking analysis (NTA). The exosomal markers CD9, CD63, and HSP70 were strongly detected. The proliferation rate of the cells in group B increased after 24h of coculture. Immunofluorescence analyses revealed that the upregulation of VEGF-A expression in HRECs stimulated by high glucose could be downregulated by cocultured hUCMSC-derived exosomes (F=39.03, P<0.01). The upregulation of VEGF-A protein (group C: F=7.96; group D: F=17.29; group E: F=11.89; 8h: F=9.45; 16h: F=12.86; 24h: F=42.28, P<0.05) and mRNA (group C: F=4.137; group D: F=13.64; group E: F=22.19; 8h: F=7.253; 16h: F=16.98; 24h: F=22.62, P<0.05) in HRECs stimulated by high glucose was downregulated by cocultured hUCMSC-derived exosomes (P<0.05). CONCLUSION: hUCMSC-derived exosomes downregulate VEGF-A expression in HRECs stimulated by high glucose in time and concentration dependent manner.

Protective effects of umbilical cord mesenchymal stem cell exosomes in a diabetic rat model through live retinal imaging.

AIM: To assess the protective effect of human umbilical cord mesenchymal stem cell exosomes (hucMSC-Exs) in a diabetic rat model by using a variety of retinal bioassays. METHODS: hucMSCs were subjected to differential ultracentrifugation for the collection of exosomes, and transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) using a NanoSight analysis system and Western blotting (WB) were used to analyze the expression of surface marker proteins such as CD63, CD9 and Calnexin. Streptozotocin (STZ) was injected into the intraperitoneal cavity to establish a diabetic model. Rats were divided into a normal group, diabetic group and hucMSC-Ex group. Fundus fluorescein angiography (FFA), optical coherence tomography (OCT) and other live imaging methods were used to observe the fundus of the rats. Finally, the eyeballs of rats from each group were collected for hematoxylin-eosin (HE) staining to further analyze the retinal structure. RESULTS: Through TEM, NTA and WB, we successfully isolated hucMSC-Exs. Subsequent FFA and OCT confirmed that hucMSC-Exs effectively prevented early retinal vascular damage and thickening of the retina. Finally, HE staining of rat retinal sections revealed that exosomes effectively alleviated retinal structure disruption caused by diabetes. CONCLUSION: hucMSC-Exs have a protective effect on the retina in diabetic rat through FFA, OCT and HE staining.

Protective effect of human umbilical cord mesenchymal stem cell-derived exosomes on rat retinal neurons in hyperglycemia through the brain-derived neurotrophic factor/TrkB pathway.

AIM: To explore whether human umbilical cord mesenchymal stem cell (hUCMSC)-derived exosomes (hUCMSC-Exos) protect rat retinal neurons in high-glucose (HG) conditions by activating the brain-derived neurotrophic factor (BDNF)-TrkB pathway. METHODS: hUCMSC-Exos were collected with differential ultracentrifugation methods and observed by transmission electron microscopy. Enzyme-linked immunosorbent assays (ELISAs) was used to quantify BDNF in hUCMSC-Exos, and Western blot was used to identify surface markers of hUCMSC-Exos. Rat retinal neurons were divided into 4 groups. Furthermore, cell viability, cell apoptosis, and TrkB protein expression were measured in retinal neurons. RESULTS: hUCMSCs and isolated hUCMSC-Exos were successfully cultured. All hUCMSC-Exos showed a diameter of 30 to 150 nm and had a phospholipid bimolecular membrane structure, as observed by transmission electron microscopy. ELISA showed the BDNF concentration of hUCMSCs-Exos was 2483.16±281.75. hUCMSCs-Exos effectively reduced the apoptosis of retinal neuron rate and improved neuron survival rate, meanwhile, the results of immunofluorescence verified the fluorescence intensity of TrKB in neurons increased. And all above effects were reduced by treated hUCMSCs-Exos with BDNF inhibitors. hUCMSC-Exos effectively reduced the apoptosis rate of retinal neurons by activating the BDNF-TrkB pathway in a HG environment. CONCLUSION: In the HG environment, hUCMSC-Exos could carry BDNF into rat retinal neurons, inhibiting neuronal apoptosis by activating the BDNF-TrkB pathway.

Protective effect of LIF-huMSCs on the retina of diabetic model rats.

AIM: To investigate the protective effect of human umbilical cord mesenchymal stem cells (hUCMSCs) modified by the LIF gene on the retinal function of diabetic model rats and preliminarily explore the possible mechanism. METHODS: A stably transfected cell line of hUCMSCs overexpressing leukemia inhibitory factor (LIF) was constructed. Overexpression was verified by fluorescent quantitative polymerase chain reaction (qPCR). Forty-eight adult Sprague-Dawley rats were randomly divided into a normal control group (group A), streptozotocin-induced diabetic control group (group B), diabetic rats at 3mo injected with empty vector-transfected hUCMSCs (group C) or injected with LIF-hUCMSCs (group D). Four weeks after the intravitreal injection, analyses in all groups included retinal function using flash electroretinogram (F-ERG), retinal blood vessel examination of retinal flat mounts perfused with fluorescein isothiocyanate-dextran (FITC-dextran), and retinal structure examination of sections using hematoxylin and eosin staining. Expression levels of adiponectin (APN), high-sensitivity C-reactive protein (hs-CRP), and neurotrophin-4 (NT-4) in each group was detected using immunohistochemistry, PCR, Western blotting, and ELISA, respectively. RESULTS: A stable transgenic cell line of LIF-hUCMSCs was constructed. F-ERG and FITC-dextran examinations revealed no abnormalities of retinal structure and function in group A, severe damage of the retinal blood vessels and function in group B, and improved retinal structure and function in group C and especially group D. qPCR, ELISA, and Western blot analyses revealed progressively higher APN and NT-4 expression levels in groups B, C, and D than in group A. hs-CRP expression was significantly higher in group B than in groups A, C, and D, and was significantly higher in group C than in group D (P<0.05). CONCLUSION: LIF-hUCMSCs protect the retina of diabetic rats by upregulating APN and NT-4 expression and downregulating hs-CRP expression in the retina.

Scutellarin improves the radiosensitivity of non-small cell lung cancer cells to iodine-125 seeds via downregulating the AKT/mTOR pathway.

BACKGROUND: In our previous study, we indicated that scutellarin (SCU) induced an anticancer effect in A549 cells. However, whether SCU regulates the radiosensitivity of non-small cell lung cancer (NSCLC) and its related mechanism is still unclear. METHODS: In this study, we explored the anticancer effect induced by iodine-125 (125 I) and SCU at a sensitizing concentration in A549 and H1975 cells. Cellular apoptosis and proliferation were detected by flow cytometry, Bcl-2/Bax expression level, cell cycle, CCK-8, and EdU staining. A tumor model using nude mice was also carried out to investigate the combined effect of 125 I and SCU in vivo. In addition, the expression level of AKT/mTOR pathway was detected to investigate whether it is linked to the anticancer effect of 125 I and SCU. RESULTS: SCU at a sensitizing concentration promoted the 125 I-induced apoptosis and antiproliferative effect in A549 and H1975 cells. Moreover, the same results were obtained in vivo. Based on our findings, the AKT/mTOR pathway was significantly downregulated after combined treatment with 125 I and SCU. CONCLUSIONS: The results of our study suggested that SCU promotes the anticancer effects induced by 125 I in NSCLC cells by downregulating the AKT/mTOR pathway and lays a foundation for future application of this combined treatment.

Enantioselective, Stereoconvergent Resolution Copolymerization of Racemic cis-Internal Epoxides and Anhydrides.

Unprecedented enantioselective resolution copolymerization of racemic cis-internal epoxides and anhydrides was mediated by dinuclear aluminum complexes with multiple chirality, affording optically active polyesters with two contiguous stereogenic centers, and the unreacted substrates in good enantioselectivity. Unexpected stereoconvergence is observed in this resolution copolymerization, where the selectivity factor for the enantioselective formation of copolymer significantly exceeds the kinetic resolution coefficient based on the unreacted epoxide at various conversions. Catalytic activity and copolymer enantioselectivity are strongly influenced by the phenolate ortho-substituents of the ligand set, as well as the axial linker and its chirality. An enantiopure binaphthol-linked bimetallic AlIII complex allows stereoconvergent access to the stereoregular semi-crystalline polyesters and a concomitant kinetic resolution of the epoxide substrates.

Efficacy of internal limiting membrane peeling for diabetic macular edema after preoperative anti-vascular endothelial growth factor injection.

AIM: To explore the efficacy of minimally invasive vitrectomy (MIV) with or without internal limiting membrane (ILM) peeling on the treatment of diabetic macular edema (DME) in proliferative diabetic retinopathy (PDR) combining with preoperative anti-vascular endothelial growth factor (anti-VEGF) injection. METHODS: Totally 132 eyes (132 patients) diagnosed PDR with DME were included between June 2015 and June 2018 in Tianjin Eye Hospital. The single MIV treatment group included 68 eyes and the MIV combined with ILM peeling group included 64 eyes. Anti-VEGF drugs were injected intravitreally 1wk before the operation and the period of follow-up was 1 to 3y. Best-corrected visual acuity (BCVA), central retinal thickness (CRT), total macular volume (TMV), macular edema (ME) severity, intraocular pressure (IOP), and complications were recorded. Prognostic factors of visual acuity following ILM peeling were analyzed. RESULTS: The BCVA was higher than preoperative values at 1, 3, 6, and 12mo after surgery in both groups (all P<0.05). At 6 and 12mo, the BCVA of the combined group was significantly higher than that of the MIV only group (0.52±0.23 vs 0.64±0.29 logMAR, P=0.011 in 6mo; 0.41±0.25 vs 0.52±0.25 logMAR, P=0.008 in 12mo). Mean CRT values postoperative were significantly lower than preoperative values in both groups from the 1st month (1mo 397.65±106.18 vs 451.94±118.88 µm in MIV only group; 388.88±108.68 vs 464.36±111.53 µm in combined group; both P<0.05) and decreased gradually. The differences between the two groups were statistically significant at 3, 6, and 12mo (P=0.004, 0.003, 0.00 respectively). The TMV was decreased from the 3rd month in the single treatment group (3mo 11.14±1.66 vs 12.20±2.09 mm3, P<0.05). At 12mo, the proportion of eyes with edema that had CRT more than 350 µm was significantly lower than before surgery (13.24% vs 77.94% in MIV only group; 1.56% vs 81.25% in combined group; both P<0.05). There was no significant difference in the recurrence incidence of macular epiretinal membrane, ME, transient IOP increase, vitreous rebleeding, or traction retinal detachment between the two groups. BCVA after ILM excision was positively correlated with the CRT and ME degree before and after surgery (r=0.430, 0.485, respectively; P<0.05). CONCLUSION: MIV combined with ILM peeling accelerates the absorption of ME, improves vision, reduces the postoperative CRT and TMV, and reduces the recurrence rate of postoperative ME.

Transfection with CXCR4 potentiates homing of mesenchymal stem cells in vitro and therapy of diabetic retinopathy in vivo.

AIM: To investigate the effect of the overexpression of C-X-C chemokine receptor type 4 (CXCR4) on homing of mesenchymal stem cells (MSCs) in vitro and therapeutic effects of diabetic retinopathy (DR) in vivo. METHODS: MSCs were infected by lentivirus constructed with CXCR4. The expression of CXCR4 was examined by immunofluorescence, Western blot, and quantitative polymerase chain reaction. CXCR4-overexpressing MSCs were cultured in vitro to evaluate their chemotaxis, migration, and apoptotic activities. CXCR4-overexpressing MSCs were intravitreally injected to observe and compare their effects in a mouse model of DR. The histological structure of DR in rats was inspected by hematoxylin and eosin staining. The expression of rhodopsin, neuron-specific enolase (NSE), and inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α was examined by Western blot and immunohistochemical analyses. RESULTS: The transduction of MSCs by lentivirus was effective, and the transduced MSCs had high expression levels of CXCR4 gene and protein. Improved migration activities were observed in CXCR4-overexpressing MSCs. Further, reduced retinal damage, upregulation of rhodopsin and NSE protein, and downregulation of inflammatory cytokines IL-6 and TNF-α were observed in CXCR4-overexpressing MSCs in vivo. CONCLUSION: The homing of MSCs can be enhanced by upregulating CXCR4 levels, possibly improving histological structures of DR. CXCR4-overexpressing MSCs can be a novel strategy for treating DR.

Mesenchymal stem cells-derived exosomes ameliorate blue light stimulation in retinal pigment epithelium cells and retinal laser injury by VEGF-dependent mechanism.

AIM: To observe the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells (hUCMSCs) on the expression of vascular endothelial growth factor-A (VEGF-A) in blue light injured human retinal pigment epithelial (RPE) cells and laser-induced choroidal neovascularization (CNV) in rats. METHODS: Exosomes were isolated from hUCMSCs and characterized by transmission electron microscope and Western blot. MSCs-derived exosomes were cultured with RPE cells exposed to blue light. The mRNA and protein expression of VEGF-A were determined by real time-polymerase chain reaction (PCR) and Western blot, respectively. Immunofluorescence assay was used for the detection of the expression level of VEGF-A. We injected different doses of MSCs-derived exosomes intravitreally to observe and compare their effects in a mouse model of laser-induced retinal injury. The histological structure of CNV in rats was inspected by hematoxylin-eosin (HE) staining and fundus fluorescein angiography. The expression of VEGF-A was detected by immunohistochemistry. RESULTS: Exosomes exhibited the typical characteristic morphology (cup-shaped) and size (diameter between 50 and 150 nm). The exosomes marker, CD63, and hUCMSCs marker, CD90, showed a robust presence. In vitro, MSCs-derived exosomes downregulated the mRNA(Exo-L: t=6.485, 7.959, 9.286; Exo-M: t=7.517, 10.170, 13.413; Exo-H: t=10.317, 12.234, 14.592, P<0.05) and protein (Exo-L: t=2.945, 4.477, 6.657; Exo-M: t=4.713, 6.421, 8.836; Exo-H: t=6.539, 12.194, 12.783; P<0.05) expression of VEGF-A in RPE cells after blue light stimulation. In vivo, we found that the MSCs-derived exosomes reduced damage, distinctly downregulated VEGF-A (Exo-H: t=0.957, 1.382; P<0.05), and gradually improved the histological structures of CNV for a better visual function (Exo-L: 0.346, Exo-M: 3.382, Exo-H: 8.571; P<0.05). CONCLUSION: MSCs-derived exosomes ameliorate blue light stimulation in RPE cells and laser-induced retinal injury via downregulation of VEGF-A.