Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: A bioengineered lymphatic vessel model for studying lymphatic endothelial cell-cell junction and barrier function

doi: 10.1111/micc.12730

Figure Lengend Snippet: (A) A schematic of an organotypic 3D lymphatic vessel model (LV-on-chip). Prox-1 (green) and CD31 (red) expression confirms lymphatic endothelial identity and cell morphology in the channel. (B) Morphologic changes in human dermal microvascular blood endothelial cells (BECs) with lymphatic endothelial cells (LECs) after one day of cell seeding. BECs become more contractile than LECs, forming a smaller vessel diameter compared to LECs. (C) BVs and LVs observed in mouse ear tissues. mLYVE-1, anti-mouse LYVE-1 antibody; mCD31, anti-mouse CD31 antibody. (D) Phalloidin (red) and anti-VE-cad (VE-cadherin) antibody (green) staining to visualize F-actin and adherens junctions. (E) Lymphatic and blood vessel barrier function. 70 kDa dextran was introduced into the vessel lumens and dextran diffusion was observed in real time under microscopy. Superimposed red dashed lines represent the edges of the vessel lumens. (F) Quantification of the permeability of BEC-generated engineered BVs and LEC-generated LVs. ** p = 0.0016, two tailed unpaired Student t-test, n = 5 per group. Data are expressed as mean ± S.E.M.

Article Snippet: In the hollow channel, we seeded human dermal microvascular lymphatic endothelial cells (LECs) to form a biomimetic lymphatic vessel ( ).

Techniques: Expressing, Staining, Diffusion-based Assay, Microscopy, Permeability, Generated, Two Tailed Test

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: A bioengineered lymphatic vessel model for studying lymphatic endothelial cell-cell junction and barrier function

doi: 10.1111/micc.12730

Figure Lengend Snippet: (A) Lymphatic endothelial cells (LECs) in different ECM hydrogels (2D): 2.5 mg/ml collagen 1, 2.5 mg/ml collagen 1 and 150 μg/ml Fibronectin, and no gel (plastic). F-actin and VE-cad were visualized to assess cytoskeletal arrangement and adherens junction formation in each condition. (B) Quantification of the relative junction area was performed, illustrating a significantly lower junction area in cells grown on the 2.5 mg/ml collagen 1 compared to the cells grown directly on plastic. ** p = 0.0017 (Collagen 1 vs. plastic); higher junction area in cells grown on the 2.5 mg/ml collagen 1 + fibronectin compared to the cells grown on collagen 1. * p = 0.0151 (Collagen 1 + fibronectin vs. Collagen 1); not-significant (ns) p = 0.5292 (Collagen 1 + fibronectin vs plastic). One-way ANOVA with Tukey’s HSD tests , n = 6 per group. Data are expressed as mean ± S.E.M. (C) Dynamics of fibronectin on LECs in collagen 1 or collagen 1 + fibronectin gel. On collagen 1 gel, LEC islands with VE-cad expression lacks fibronectin expression. On collagen 1 + fibronectin, fibronectin connects separate LEC islands. (D) At day 4 on Collagen 1 + fibronectin, LECs showed tightened junctions and fibronectin was localized in the junctional area.

Article Snippet: In the hollow channel, we seeded human dermal microvascular lymphatic endothelial cells (LECs) to form a biomimetic lymphatic vessel ( ).

Techniques: Expressing

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: A bioengineered lymphatic vessel model for studying lymphatic endothelial cell-cell junction and barrier function

doi: 10.1111/micc.12730

Figure Lengend Snippet: (A) Activated integrin α5 was visualized in both ECM composition conditions by using anti-integrin α5 antibody (clone: SNAKA51) that can only detect the activated form of the integrin α5. F-actin was also observed in these conditions. (B) LECs in Collagen 1 were pre-treated with anti-integrin α5 antibodies (clone: SNAKA51) antibodies to activate integrin α5 in LECs. The fixed samples were stained with anti-VE-cadherin antibodies, anti-JAM-A antibodies, and phalloidin to visualize adherens junctions and F-actin. (C) Quantification of the relative junction area was performed, illustrating a significantly higher junction area in integrin α5 activated cells compared to the control LECs. ** p = 0.0020; Two tailed unpaired Student t-test, n = 6 per group. Data are expressed as mean ± S.E.M. (D) Control LECs or LECs with activated integrin α5 were seeded in LV-on-chip and cultured for 3 days on the rocking platform. 70 kDa dextran was introduced to the lymphatic lumens. Dextran diffusion was observed at 0 and 1 minutes under microscopy. Superimposed red dashed lines represent the edges of the vessel lumens. (E) Quantification of the permeability of LEC-generated engineered LVs in collagen 1 with and without integrin α5 activation. ** p = 0.0021. Two tailed unpaired Student t-test, n = 5 per group. Data are expressed as mean ± S.E.M. (F) This table summarizes our findings regarding LEC permeability and integrin α5 activity. LVs grown in Collagen 1 without any activator treatment showed high LEC permeability and low integrin α5 activity. In contrast, LVs grown in either Collagen 1 + Fibronectin or LVs grown in only Collagen 1 with integrin α5 activator pre-treatment both showed low LEC permeability and high integrin α5 activity.

Article Snippet: In the hollow channel, we seeded human dermal microvascular lymphatic endothelial cells (LECs) to form a biomimetic lymphatic vessel ( ).

Techniques: Staining, Control, Two Tailed Test, Cell Culture, Diffusion-based Assay, Microscopy, Permeability, Generated, Activation Assay, Activity Assay

Journal: Scientific Reports

Article Title: Hindlimb Ischemia Impairs Endothelial Recovery and Increases Neointimal Proliferation in the Carotid Artery

doi: 10.1038/s41598-017-19136-6

Figure Lengend Snippet: Effects of hindlimb ischemia on carotid artery endothelium. ( a ) Left: Representative sections of carotid arteries immunostained for the specific endothelial cell marker CD31. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) in carotid arteries sections explanted from BI and FL-BI groups at 14 days after balloon injury. Scale bars = 50 µm. Right: Bar graphs represents the percentage of re-endothelializated circumference of the common carotid artery. * P < 0.05 versus BI group, n = 6 for group. ( b ) Schematic model of the experimental setup. ( c ) Expression levels of selected miRNAs in the carotid artery endothelium 14 days from injury. * P < 0.05 versus BI group; n = 6. ( d ) Relative expression of eNOS, VCAM and ICAM mRNA transcripts in carotid artery endothelium 14 days after injury. * P < 0.05 versus BI group; n = 6.

Article Snippet: Endothelial repair in balloon-injured arteries was evaluated at 14 days after vascular injury using a primary antibody against the endothelial cell surface marker CD31 (R&D Systems, catalog #AF3628, 1:50 dilution).

Techniques: Marker, Staining, Expressing

Journal: Scientific Reports

Article Title: Hindlimb Ischemia Impairs Endothelial Recovery and Increases Neointimal Proliferation in the Carotid Artery

doi: 10.1038/s41598-017-19136-6

Figure Lengend Snippet: Systemic delivery of antagomiR-16 promotes endothelial recovery and inhibits neointima formation in the carotid artery of rats with hindlimb ischemia. ( a ) Schematic model of the experimental setup. ( b ) Expression levels of miR-16 in vascular endothelium. Total RNAs were obtained from vascular endothelium of rat carotid artery 14 days after injury. * P < 0.01 versus control group; n = 6. ( c ) Relative expression of RhoGDIα and eNOS mRNA transcripts in vascular endothelium of rat carotid artery 14 days after injury. * P < 0.05 versus control group; n = 6. ( d ) Left: Representative images of Haematoxylin and eosin staining in balloon-injured carotid arteries at 14 days in rats treated with or without Antago-16. Scale bars, 100 µm. Right: Bar graphs represent the morphometric analysis of arterial sections. Neointima /media ratio of arteries in differently treated groups is shown. * P < 0.05 versus NC group; n = 7. ( e ) Left: Representative sections of carotid arteries immunostained for the specific endothelial cell marker CD31. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Carotid arteries were explanted from experimental groups at 14 days after balloon injury. Scale bars = 50 µm. Right: Bar graphs represents the percentage of re-endothelializated circumference of the common carotid artery. * P < 0.05 versus rat treated with antagomir scrambled; n = 6 for group. ( f ) Left: Representative sections of carotid arteries stained for the macrophage (brown) marker CD68. Carotid arteries were explanted from rats at 14 days after balloon injury. Right: Quantitative data derived from arterial sections stained with CD68 positive cells. * P < 0.05 versus control; n = 5.

Article Snippet: Endothelial repair in balloon-injured arteries was evaluated at 14 days after vascular injury using a primary antibody against the endothelial cell surface marker CD31 (R&D Systems, catalog #AF3628, 1:50 dilution).

Techniques: Expressing, Control, Staining, Marker, Derivative Assay

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: Diabetes

Article Title: Decreased Cerebrovascular Brain-Derived Neurotrophic Factor–Mediated Neuroprotection in the Diabetic Brain

doi: 10.2337/db10-1371

Figure Lengend Snippet: BDNF expression is reduced in the diabetic brain endothelium. Representative images of cortical sections (2-mm thick) from 6-week diabetic and age-matched nondiabetic rats immunostained for BDNF. Blood vessels (arrows) are visualized by CD31 staining ( left ). Cortex microvessels from a diabetic rat show decreased BDNF immunofluorescence when compared with those of a nondiabetic rat ( right ). Magnification 40×. (A high-quality digital representation of this figure is available in the online issue.)

Article Snippet: Primary human brain microvessel endothelial cells were purchased from Cell Systems Corporation (Kirkland, WA), mostly derived from a heterogenous mix of rapidly autopsied human brains obtained within a few hours after death.

Techniques: Expressing, Staining, Immunofluorescence

Journal:

Article Title: Divalent ion block of inward rectifier current in human capillary endothelial cells and effects on resting membrane potential

doi: 10.1111/j.1469-7793.1998.119bf.x

Figure Lengend Snippet: A, current traces from a HCEC recorded in response to voltage step pulses from −120 to +10 mV in 20 mV intervals at a holding potential of −60 mV. B, current traces from the same voltage pulse protocol after application of 50 μm Ba2+. C, current-voltage plot of peak (•) and steady-state (▪) current in standard bath solution, and peak (○) and steady-state (□) current after application of Ba2+ for the same cell. D, current traces from a HCEC response to voltage steps of −120 mV to +10 mV from a holding potential of −60 mV with 5.4 mm K+ in the standard bath solution. E, current traces from the same voltage protocol in the absence of external K+. F, peak current-voltage plot from the currents in D and E before (•) and after (○) removing all external K+. Elimination of external K+ completely blocks the inward current.

Article Snippet: Cell culture Human capillary endothelial cells (HCEC) were obtained from Cell Systems Corporation (WA, USA), and were cultured in growth medium for human capillary endothelial cells (4M1-500, Cell Systems Corporation).

Techniques:

Journal:

Article Title: Divalent ion block of inward rectifier current in human capillary endothelial cells and effects on resting membrane potential

doi: 10.1111/j.1469-7793.1998.119bf.x

Figure Lengend Snippet: A, inward rectifier current traces of a HCEC bathed in standard bath solution. The cell was held at −60 mV and step potentials were applied from −120 to +20 mV, in 20 mV steps. B, the same cell after superfusion with standard bath solution containing 9 mm Ca2+. C, addition of 50 μm Ba2+ blocked the remaining current. D, current traces taken after washout of high external Ca2+ and Ba2+ with standard bath solution. E, current-voltage plots for control, 9 mm Ca2+, 50 μm Ba2+ and after washout.

Article Snippet: Cell culture Human capillary endothelial cells (HCEC) were obtained from Cell Systems Corporation (WA, USA), and were cultured in growth medium for human capillary endothelial cells (4M1-500, Cell Systems Corporation).

Techniques: Control

Journal:

Article Title: Divalent ion block of inward rectifier current in human capillary endothelial cells and effects on resting membrane potential

doi: 10.1111/j.1469-7793.1998.119bf.x

Figure Lengend Snippet: A, effect of [Ca2+]o from 0.5 to 30 mm on the mean percentage block of peak inward rectifier current at −120 mV (n = 5). Inhibition of the current at different concentrations of external Ca2+ is well fitted by a logistic equation with an EC50 value of 5.4 ± 0.6 mm. B, representative current traces of a HCEC at −120 mV with [Ca2+]o from 0 to 9 mm. C, shifts in zero current level of a HCEC exposed to divalent cations. The cell was held at −60 mV and voltage ramps were applied from −120 to +20 mV. The zero current level occurred at −62 mV with 0.5 mm Ca2+, −47 mV with 7 mm Ca2+, and −18 mV with 50 μm Ba2+ in the bath solution.

Article Snippet: Cell culture Human capillary endothelial cells (HCEC) were obtained from Cell Systems Corporation (WA, USA), and were cultured in growth medium for human capillary endothelial cells (4M1-500, Cell Systems Corporation).

Techniques: Blocking Assay, Inhibition

Journal:

Article Title: Divalent ion block of inward rectifier current in human capillary endothelial cells and effects on resting membrane potential

doi: 10.1111/j.1469-7793.1998.119bf.x

Figure Lengend Snippet: A, current traces from a HCEC in Mn2+-free standard bath solution. The cell was held at −60 mV and pulsed from −120 mV to +20 mV in 20 mV steps. B, current traces after switching to a bath solution containing 5 mm Mn2+. C, the current-voltage plot of the same cell in standard bath solution and in bath solution containing 5 mm Mn2+. D, current traces (same voltage protocol as A) of another HCEC in standard bath solution without Mg2+. E, current traces after application of 5 mm Mg2+ in the standard bath solution to the same HCEC. F, plot of the current-voltage relationship of the HCEC before and after application of Mg2+.

Article Snippet: Cell culture Human capillary endothelial cells (HCEC) were obtained from Cell Systems Corporation (WA, USA), and were cultured in growth medium for human capillary endothelial cells (4M1-500, Cell Systems Corporation).

Techniques:

Journal:

Article Title: Divalent ion block of inward rectifier current in human capillary endothelial cells and effects on resting membrane potential

doi: 10.1111/j.1469-7793.1998.119bf.x

Figure Lengend Snippet: A, IK(IR) traces during strong hyperpolarizing pulses (−180 to +20 mV) from a holding potential of −60 mV from a HCEC perfused with standard external solution. B, current traces from the same cell in the presence of 5 mm Sr2+. Both the transient and steady-state components of the inward current were blocked. C, current-voltage plot for the peak current (▪) and the current at the end of the pulse (•) in standard bath solution, and the peak current (□) and the steady-state current (○) in 5 mm Sr2+.

Article Snippet: Cell culture Human capillary endothelial cells (HCEC) were obtained from Cell Systems Corporation (WA, USA), and were cultured in growth medium for human capillary endothelial cells (4M1-500, Cell Systems Corporation).

Techniques: