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

Journal: Pharmacology Research & Perspectives

Article Title: Cilostazol prevents retinal ischemic damage partly via inhibition of tumor necrosis factor-α-induced nuclear factor-kappa B/activator protein-1 signaling pathway

doi: 10.1002/prp2.6

Figure Lengend Snippet: Effects of cilostazol on tumor necrosis factor-α (TNF-α)-induced disruption of tight junction proteins in the human retinal microvascular endothelial cells (HRMECs). (A, C) Representative band images for (A) zonula occludens 1 (ZO-1) and (C) claudin-5 in HRMECs. (B, D) Quantitative analysis of the band densities for ZO-1 (B) and claudin-5 (D). Data are shown as means ± SEM ( n = 3 or 4). # P < 0.05 versus control; * P < 0.05 versus vehicle.

Article Snippet: Primary human retinal microvascular endothelial cells (HRMECs) were obtained from DS Pharma Biomedical (Osaka, Japan).

Techniques:

Journal: Experimental eye research

Article Title: Inhibition of interleukin-6 trans-signaling prevents inflammation and endothelial barrier disruption in retinal endothelial cells.

doi: 10.1016/j.exer.2018.09.009

Figure Lengend Snippet: Western blot analysis was conducted in human retinal endothelial cells (HREC) to detect glycoprotein 130 (gp130) and IL6 receptor expression. (A) HREC express sgp130 (B) but lack the membrane bound IL6 receptor (IL6R). The positive control used were hepatocyte (HepG2) cells which expresses IL6R. β-actin served as a loading control. (C) HRECs were treated with and without sgp130Fc (10µg/mL) for 60 min followed by IL-6 (10 ng/ml) alone or combined with sIL-6R (150 ng/ml) for 10 min. Cells were collected and stained using monoclonal antibody anti-mouse p-STAT3 (pY705) (Millipore) and FITC-conjugated anti-mouse AlexaFluor 488 Conjugate (Cell Signaling) and analyzed using the FACS Calibur flow cytometer. (D) IL-6 alone did not show any response in HRECs but sgp130Fc pre-treatment, prevented STAT3 phosphorylation observed in IL-6+sIL-6R treated cells. Bar graphs represent fold untreated of peak means. Results are expressed as means ± SEM; n = 4 per group, * p < 0.05 versus IL- 6+sIL-6R; † p < 0.05 versus IL-6+sIL-6R+sgp130 Fc.

Article Snippet: Human retinal endothelial cells (Lonza, USA) were maintained on gelatin-coated culture dishes in M199 supplemented media with 10% fetal bovine serum (Hyclone Logan, UT, USA), 10% MCDB-131 (VecTech Rensselaer, NY, USA), and 1% L-glutamine/penicillin/ streptomycin (Gemini West Sacramento, CA, USA) at 37°C in a humi dified incubator containing 5% CO 2 with the media changed every alternate day.

Techniques: Western Blot, Expressing, Membrane, Positive Control, Control, Staining, Flow Cytometry, Phospho-proteomics

Journal: Experimental eye research

Article Title: Inhibition of interleukin-6 trans-signaling prevents inflammation and endothelial barrier disruption in retinal endothelial cells.

doi: 10.1016/j.exer.2018.09.009

Figure Lengend Snippet: HRECs were treated with and without sgp130Fc (10µg/mL, 60 min) and exposed to IL-6 (10ng/mL) and sIL-6R (150ng/mL) overnight. Trans-endothelial electrical resistance (TER) was measured using an electric cell impedance sensing (ECIS) apparatus. IL-6 trans-signaling significantly disrupted endothelial barrier function. Pre- treatment with sgp130Fc maintained the endothelial barrier function at near control levels. Results are expressed as means ± SEM; n = 3 per group, * p < 0.05 vs untreated; † p < 0.05 vs IL-6+sIL-6R.

Article Snippet: Human retinal endothelial cells (Lonza, USA) were maintained on gelatin-coated culture dishes in M199 supplemented media with 10% fetal bovine serum (Hyclone Logan, UT, USA), 10% MCDB-131 (VecTech Rensselaer, NY, USA), and 1% L-glutamine/penicillin/ streptomycin (Gemini West Sacramento, CA, USA) at 37°C in a humi dified incubator containing 5% CO 2 with the media changed every alternate day.

Techniques: Control