Journal: Journal of Diabetes Investigation

Article Title: Long non‐coding ribonucleic acid ATP2B1‐AS1 modulates endothelial permeability through regulating the miR‐4729–IQGAP2 axis in diabetic retinopathy

doi: 10.1111/jdi.13740

Figure Lengend Snippet: Identification of ATPase plasma membrane Ca 2+ transporting 1 antisense ribonucleic acid 1 (ATP2B1‐AS1) in diabetic retinopathy (DR) and high‐glucose‐treated high‐glucose‐treated human retinal endothelial cells (HRECs). (a) The heatmap of the differentially expressed genes in low glucose (LG) and high glucose (HG). Upregulated genes and downregulated genes are shown in red and blue. (b) Volcano plots showing long non‐coding ribonucleic acids expression in the LG and HG groups. The red dots show the significant expressed genes. (c) Reverse transcription quantitative polymerase chain reaction was carried out to detect ATP2B1‐AS1 levels in 5 mmol/L or 25 mmol/L glucose treated HRECs. (d) Reverse transcription quantitative polymerase was carried out to distinguish the level of ATP2B1‐AS1 in blood samples obtained from DR patients ( n = 30) and healthy individuals. All values were represented by the mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human retinal endothelial cells (HRECs) and 293T cells were purchased from ScienCell Research Laboratories (San Diego, CA, USA).

Techniques: Clinical Proteomics, Membrane, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Journal of Diabetes Investigation

Article Title: Long non‐coding ribonucleic acid ATP2B1‐AS1 modulates endothelial permeability through regulating the miR‐4729–IQGAP2 axis in diabetic retinopathy

doi: 10.1111/jdi.13740

Figure Lengend Snippet: ATPase plasma membrane Ca 2+ transporting 1 antisense ribonucleic acid 1 (ATP2B1‐AS1) prevents cell proliferation, migration, angiogenesis and permeability. (a) Reverse transcription quantitative polymerase chain reaction was made to measure the expression of ATP2B1‐AS1 after transfecting plasmid cloning deoxyribonucleic acid (pcDNA)‐long non‐coding ribonucleic acids (lncRNA) ATP2B1‐AS1 (pcDNA‐lnc) and (short hairpin RNA‐lncRNA ATP2B1‐AS1; shR‐lnc) into high‐glucose‐treated human retinal endothelial cells (HRECs). (b) The level of ATP2B1‐AS1 was detected by reverse transcription polymerase chain reaction after transfecting pcDNA‐lnc and shR‐lnc into HRECs by Cell Counting Kit‐8 assay. (c) Proliferation of HRECs was detected by Cell Counting Kit‐8 assay. (d, e) Migration ability was measured by wound healing migration assay and transwell assay. (f) Tube formation assay was used to distinguish angiogenesis ability in HRECs. (g) Cell junctional assembly formation of CDH5 staining. (h) Vascular permeability was detected by using evans blue injection. All values were represented by the mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human retinal endothelial cells (HRECs) and 293T cells were purchased from ScienCell Research Laboratories (San Diego, CA, USA).

Techniques: Clinical Proteomics, Membrane, Migration, Permeability, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation, Cloning, shRNA, Polymerase Chain Reaction, Cell Counting, Transwell Assay, Tube Formation Assay, Staining, Injection, Standard Deviation

Journal: Journal of Diabetes Investigation

Article Title: Long non‐coding ribonucleic acid ATP2B1‐AS1 modulates endothelial permeability through regulating the miR‐4729–IQGAP2 axis in diabetic retinopathy

doi: 10.1111/jdi.13740

Figure Lengend Snippet: ATPase plasma membrane Ca 2+ transporting 1 antisense ribonucleic acid 1 (ATP2B1‐AS1) sponges microRNA (miR)‐4729. The microRNAs lists and scores on predicted by using the MicroRNA Target Prediction Database. (b) Predicted miR‐4729 binding sites in 3′UTR of ATP2B1‐AS1 and dual luciferase report assay in ATP2B1‐AS1‐wild type (WT) or ATP2B1‐AS1‐mutation (MUT) co‐transfected with miR negative control (NC) or miR‐4729 mimics. (c) Level of miR‐4729 in high‐glucose‐treated human retinal endothelial cells (HRECs) transfected with shR‐lnc or pcDNA‐lnc. (d) miR‐4729 expression in blood from diabetes retinopathy (DR) patients ( n = 30) and non‐DR individuals. (e) Pearson's correlation analysis was used to check the relationship between ATP2B1‐AS1 and miR‐4729. All values were represented by the mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human retinal endothelial cells (HRECs) and 293T cells were purchased from ScienCell Research Laboratories (San Diego, CA, USA).

Techniques: Clinical Proteomics, Membrane, Binding Assay, Luciferase, Mutagenesis, Transfection, Negative Control, Expressing, Standard Deviation

Journal: Journal of Diabetes Investigation

Article Title: Long non‐coding ribonucleic acid ATP2B1‐AS1 modulates endothelial permeability through regulating the miR‐4729–IQGAP2 axis in diabetic retinopathy

doi: 10.1111/jdi.13740

Figure Lengend Snippet: ATPase plasma membrane Ca 2+ transporting 1 antisense ribonucleic acid 1 (ATP2B1‐AS1) reduced high glucose‐treated high‐glucose‐treated human retinal endothelial cells (HRECs) cell proliferation, migration, angiogenesis and permeability through regulating microRNA (miR)‐4729–IQ motif‐containing GTPase‐activating protein 2 (IQGAP2) axis. (a) Schematic indicating the miR‐4729 sites in IQGAP2 and dual luciferase assay in IQGAP2‐wild type (WT) or IQGAP2‐mutation (MUT) treated HRECs co‐transfected with miR‐NC or miR‐4729 mimics. (b) The protein IQGAP2 level was detected by WB after transfection. (c) HRECs proliferation was detected by Cell Counting Kit‐8 assay after transfection. (d, e) Migration ability was measured by wound healing migration assay and transwell assay after transfection. (f) Tube formation assay was used to detect the ability of angiogenesis in HRECs after transfection. (g) Cell junctional assembly formation of VE‐cadherin staining after transfection. All values were represented by the mean ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Human retinal endothelial cells (HRECs) and 293T cells were purchased from ScienCell Research Laboratories (San Diego, CA, USA).

Techniques: Clinical Proteomics, Membrane, Migration, Permeability, Luciferase, Mutagenesis, Transfection, Cell Counting, Transwell Assay, Tube Formation Assay, Staining, Standard Deviation

Journal: Regenerative engineering and translational medicine

Article Title: CD140b (PDGFRβ) signaling in adipose-derived stem cells mediates angiogenic behavior of retinal endothelial cells

doi: 10.1007/s40883-018-0068-9

Figure Lengend Snippet: Representative images of HREs co-cultured with control siRNA treated ASCs or CD140b siRNA treated ASCs for 6 days. (A) Upper panel shows colored images of HREs, stained with Isolectin B4 (red); ASCs, stained for α-SMA (green), and co-cultures counter-stained with DAPI (blue). Lower panel shows Red-only channels representing angiogenic tubes stained with Isolectin B4 (4× magnification). (B) Representative high magnification images of ASCs and HREs co-culture. (C) Image analysis of vascular tube length calculated by image J software as pixels/field. Data represent Mean ± SEM performed in triplicates. *, p<0.05 using unpaired Student T-test; n=3 donors.

Article Snippet: Co-culture of ASCs and human retinal endothelial cells (HREs) Human Retinal Endothelial Cells (HREs; ACBRI 181, Cell Systems Corporation, Kirkland, WA) were co-cultured with Cd140b+ or CD140b− ASCs as described previously ( 8 , 15 ).

Techniques: Cell Culture, Control, Staining, Co-Culture Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Macrophage-Myofibroblast Transition Contributes to Myofibroblast Formation in Proliferative Vitreoretinal Disorders

doi: 10.3390/ijms241713510

Figure Lengend Snippet: Identification of CD206 + cells in epiretinal membranes. Immunohistochemical staining for the M2 macrophage marker CD206 showing immunoreactivity in ( A ) vascular endothelial cells (arrows), ( B ) stromal monocytes/macrophages (arrows) and ( C ) stromal spindle-shaped cells (arrows) in membranes from patients with proliferative diabetic retinopathy (PDR). ( D ) CD206 surface expression was detected in human retinal microvascular endothelial cells (HRMECs) using flow cytometry. Results are presented as the mean percent of CD206 + cells ± SEM and are derived from four independent experiments. Immunoreactivity for CD206 was detected ( E ) in monocytes/macrophages (arrows) and ( F ) spindle-shaped cells (arrows) in membranes from patients with proliferative vitreoretinopathy (PVR). Note that some of the CD206 + cells in the PVR membrane contained pigment. Representative figures are provided for 1 patient out of a total of 12 PDR or 12 PVR patients. Each panel is from a different patient (scale bar, 10 µm).

Article Snippet: Human retinal Müller glial cells (MIO-M1) (a generous gift from Prof. A. Limb, Institute of Ophthalmology, University College, London, UK) and human retinal microvascular endothelial cells (HRMECs) (Cell Systems Corporation, Kirkland, WA, USA) were cultured as described previously [ ].

Techniques: Immunohistochemical staining, Staining, Marker, Expressing, Flow Cytometry, Derivative Assay, Membrane

Journal: Biosensors

Article Title: In Vitro Wounding Models Using the Electric Cell-Substrate Impedance Sensing (ECIS)-Zθ Technology

doi: 10.3390/bios8040090

Figure Lengend Snippet: Changes in the hCMVEC morphology after wounding on the 8W10E+ and 8W1E arrays using the ECIS-Zθ system. Expression of the adherens junction protein, VE-cadherin, and the tight junction regulating protein, ZO-1, under control and wounded conditions in the hCMVECs on the 8W1E and 8W10E+ arrays. Representative images of three independent experiments are shown; images are a Z-stack composition between 0.4–0.8 μm at 48 h post-wounding and the control cells. The hCMVECs are labelled for VE-cadherin using mouse monoclonal CD144 antibody, ZO-1 using mouse monoclonal ZO-1 antibody, visualized by goat α-mouse Alexa Fluor 488 (green) at 40× magnification on the LSM 710 inverted confocal microscope. Actin filaments are stained with ActinRed 555 ReadyProbes Reagent (red). Nuclei are counterstained with Hoechst (blue). The hCMVECs were seeded at a density of 60,000 cells/cm 2 . A wounding current of 3000 uA at 60 kHz was delivered for 30 s to selected wells on the 8W1E array, and a wounding current of 5000 uA at 60 kHz was delivered for 60 s to selected wells on the 8W10E+ array. Scale bar = 50 μm.

Article Snippet: The human cerebral microvascular endothelial (hCMVEC) cell line was purchased from Applied Biological Materials (cat#T0259, ABM Good, Richmond, BC, Canada), and has been extensively characterized in terms of junctional protein expression and transendothelial electrical resistance (TEER) (O’Carroll et al., 2015; Wiltshire et al., 2016).

Techniques: Expressing, Control, Microscopy, Staining