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: PLoS ONE

Article Title: Ras/MAPK Signaling Modulates VEGFR-3 Expression through Ets-Mediated p300 Recruitment and Histone Acetylation on the Vegfr3 Gene in Lymphatic Endothelial Cells

doi: 10.1371/journal.pone.0051639

Figure Lengend Snippet: A. Western blots of phosphorylated Ets1 (pEts1), Ets1 and total Ras proteins in immortalized mouse LECs (mLECs) transfected with control siRNA or mixed siRNAs for ras genes ( Hras , Nras and Kras ) in the presence or absence of VEGF-A, VEGF-C or VEGF-D. B. Real-time RT-PCR assay for mRNAs in mLECs transfected with control, Ets1 and Ets2 siRNAs. si-1 and -2 represent two individual siRNAs. Ets1/2 si represents transfection with mixed siRNAs for Ets1 and Ets2. Error bars represent the S.D.; n = 3. *p<0.05, ***p<0.005, ****p<0.001 (vs. mLECs transfected with control siRNA; see and ). C. Protein expression in mLECs transfected with control, Ets1 and Ets2 siRNAs. Left panel, western blots; right panel, quantitative analysis of western blots. Error bars represent the S.D.; n = 3. **p<0.01, ***p<0.005, ****p<0.001 (vs. mLECs transfected with control siRNA).

Article Snippet: Human dermal microvascular LECs (hLECs) from pooled donors (Lonza) were cultured on gelatinized culture dishes using an EGM-2 MV bullet kit (Lonza) according to the manufacturer’s protocol.

Techniques: Western Blot, Transfection, Control, Quantitative RT-PCR, Expressing

Journal: PLoS ONE

Article Title: Ras/MAPK Signaling Modulates VEGFR-3 Expression through Ets-Mediated p300 Recruitment and Histone Acetylation on the Vegfr3 Gene in Lymphatic Endothelial Cells

doi: 10.1371/journal.pone.0051639

Figure Lengend Snippet: A. VEGFR-3 and Gapdh protein expression in Vegfr3-knockdown LECs. Upper panel, western blots; lower panel, quantitative analysis of western blots. Error bars represent the S.D.; n = 3. ****p<0.001 (vs. mLECs transfected with control siRNA). B. WST-1 assays using Vegfr3-knockdown mLECs (upper left panel) and Ets-knockdown mLECs (lower left panel), and BrdU assays using Vegfr3-knockdown mLECs (upper right panel) and Ets-knockdown mLECs (lower right panel). Error bars represent the S.D.; n = 12. *p<0.05, ****p<0.001 (vs. mLECs transfected with control siRNA in each assay). C. DiI-stained cellular networks of mLECs transfected with control, Ets1, Ets2, and Vegfr3 siRNAs on Matrigel. Scale bar = 500 µm. DiI-labeled areas were quantified and the mean area of DiI-labeled wild-type mLECs was normalized to 1. Error bars represent the S.D.; n = 3. **p<0.01, ***p<0.005, ****p<0.001 (vs. mLECs transfected with control siRNA).

Article Snippet: Human dermal microvascular LECs (hLECs) from pooled donors (Lonza) were cultured on gelatinized culture dishes using an EGM-2 MV bullet kit (Lonza) according to the manufacturer’s protocol.

Techniques: Expressing, Knockdown, Western Blot, Transfection, Control, Staining, Labeling

Journal: PLoS ONE

Article Title: Ras/MAPK Signaling Modulates VEGFR-3 Expression through Ets-Mediated p300 Recruitment and Histone Acetylation on the Vegfr3 Gene in Lymphatic Endothelial Cells

doi: 10.1371/journal.pone.0051639

Figure Lengend Snippet: A. Real-time RT-PCR assay for VEGFR3 mRNA in primary human LECs (hLECs) transfected with control and ETS1 siRNAs. si-1 and -2 represent two individual siRNAs. Error bars represent the S.D.; n = 3. ***p<0.005, ****p<0.001 (vs. hLECs transfected with control siRNA; see ). B. WST-1 assays and BrdU assays using ETS1-knockdown hLECs. Error bars represent the S.D.; n = 12. ***p<0.005, ****p<0.001 (vs. hLECs transfected with control siRNA). C. DiI-stained cellular networks of hLECs transfected with control and ETS1 siRNAs on Matrigel. Scale bar = 500 µm. DiI-labeled areas were quantified and the mean area of DiI-labeled wild-type hLECs was normalized to 1. Error bars represent the S.D.; n = 3. *p<0.05, ****p<0.001 (vs. hLECs transfected with control siRNA).

Article Snippet: Human dermal microvascular LECs (hLECs) from pooled donors (Lonza) were cultured on gelatinized culture dishes using an EGM-2 MV bullet kit (Lonza) according to the manufacturer’s protocol.

Techniques: Quantitative RT-PCR, Transfection, Control, Knockdown, Staining, Labeling

Journal: PLoS ONE

Article Title: Ras/MAPK Signaling Modulates VEGFR-3 Expression through Ets-Mediated p300 Recruitment and Histone Acetylation on the Vegfr3 Gene in Lymphatic Endothelial Cells

doi: 10.1371/journal.pone.0051639

Figure Lengend Snippet: The Ras/MAPK/Ets pathway involved in transcriptional regulation of the Vegfr3 gene in LECs. Ras/MAPK signal-activated Ets proteins, together with p300, regulate Vegfr3 gene expression and lead to changes in cellular proliferation and morphogenesis.

Article Snippet: Human dermal microvascular LECs (hLECs) from pooled donors (Lonza) were cultured on gelatinized culture dishes using an EGM-2 MV bullet kit (Lonza) according to the manufacturer’s protocol.

Techniques: Gene Expression

Journal: The ocular surface

Article Title: Aqueous humor induces lymphatic regression on the ocular surface

doi: 10.1016/j.jtos.2020.03.002

Figure Lengend Snippet: Aqueous humor induced the regression of lymphatic tubes formed by human microdermal lymphatic endothelial cells in vitro. (A) Representative images showing lymphatic meshes 5 hours after culturing with AH or control medium, respectively. Scale bar: 100 μm. Images showing the sites of regression, as indicated by the arrows, are presented in (B). Scale bar: 20 μm. (C) Summarized data assessed in the number and mean size of the meshes, respectively. Assays were performed in triplicates and repeated once. *p < 0.05.

Article Snippet: Briefly, human neonatal microdermal LECs were purchased from AngioBio (AngioBio Co., Del Mar, CA, USA), and maintained in EBM MV2 growth medium (Promocell, Heidelberg, Germany), supplemented with 10% bovine calf serum (HyClone, Logan, Utah) and 1% penicillin/streptomycin.

Techniques: In Vitro, Control

Journal: Blood

Article Title: IL-7-producing stromal cells are critical for lymph node remodeling

doi: 10.1182/blood-2012-03-416859

Figure Lengend Snippet: Assessment of IL-7 expression in human LN LECs. (A) Fetal LN sections were stained with fluorescently labeled antibodies against LyveI, MadCAM-1, and CD3. (B) Adult LN sections were stained with antibodies against LyveI, VCAM-1, MadCAM-1, and Pdpn. LyveI+Pdpn+ LECs (blue in left panel and green in right panel) are indicated in the subcapsular sinus (arrows) and LN medulla (*). (C) CD31+Pdpn+ LECs, CD31−Pdpn+ FRCs, and CD31+Pdpn− BECs from human fetal mesenteric LNs were sorted by flow cytometry. IL-7 expression levels were determined by quantitative RT-PCR. Values indicate mean ± SEM from triplicates, representative results from 1 of 2 independent sorting experiments. (D) Human LN LECs were cultivated and analyzed for IL-7 mRNA expression by quantitative RT-PCR. Cell culture supernatants were collected after 48 hours and analyzed for IL-7 protein by ELISA (right graph). Measurements were carried out in triplicates (mean ± SEM). (E) MACS-isolated human naive CD4+ T cells (2 × 105) were cocultured with human LECs, HUVECs, or supernatant from human LEC cultures in the presence and absence of neutralizing anti–IL-7 antibody. After 48 hours, T-cell survival was analyzed by flow cytometry and is displayed as difference (Δ) to medium control. Values indicate mean ± SEM from triplicates, representative results from 1 of 2 independent experiments; *P < .05.

Article Snippet: Primary human LECs were obtained from ScienCell.

Techniques: Expressing, Staining, Labeling, Flow Cytometry, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, Isolation, Control