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: 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: Angiogenesis

Article Title: A novel lymphatic pattern promotes metastasis of cervical cancer in a hypoxic tumour-associated macrophage-dependent manner

doi: 10.1007/s10456-020-09766-2

Figure Lengend Snippet: Hypoxic TAMs promote lymphangiogenesis and LVEM formation. a The function of CM from different macrophage-treated HDLECs on M2-polarized THP-1 macrophages and tumour cells (SiHa) was detected by transwell array in vitro. b Statistical analysis showing the length of tube formation in vitro. Average length of tubes per field were calculated. c Statistical analysis showing the expression of LV and LVEM in mouse footpad tumour. d Representative micrographs showing tube formation assay in vitro of HDLECs pretreated with different macrophage CM for 48 h. Scale bar, 50 μm. e – i Popliteal lymphatic metastasis model was established in female C57BL/6 mice by inoculating the footpad with TC-1 cells (5 × 10 6 ). When footpad tumour size reached 50 mm 3 , macrophage supernatants of different treatment conditions (10 μl) were then injected into the centre of the tumours ( n = 5/group, repeated twice) for 2 weeks daily. After 2 weeks of induction, primary tumours reached a comparable size of ~ 150 mm 3 , and then footpad tumours and popliteal LNs were collected for study. e Representative images of LYVE-1 + lymphatic vessel (red), CD206 + TAMs (green) and DAPI (blue) fluorescence staining in footpad tumour. Images are shown at × 400 magnification (Scale bar, 50 μm). f IHC Staining of CK7 in popliteal LNs. Representative micrographs are shown (Scale bar, 100 μm). Metastasis-positive LNs were identified by staining for epithelial marker CK7. g Photos of mouse popliteal LNs in different macrophage CM-primed tumour ( n = 5/group). h Statistical analysis showing the volume (mm 3 ) of the LNs. i The ratio of metastasis-positive to total dissected popliteal LNs from mice treated with different macrophage supernatants. Error bars represent the mean ± SD of three independent experiments. * P < 0.05. N: Normoxia; H: Hypoxia

Article Snippet: Human dermal LECs (HDLECs) were purchased from ScienCell and cultured in endothelial cell medium (ScienCell, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum and endothelial growth medium supplements (ScienCell).

Techniques: In Vitro, Expressing, Tube Formation Assay, Injection, Fluorescence, Staining, Immunohistochemistry, Marker

Journal: Angiogenesis

Article Title: A novel lymphatic pattern promotes metastasis of cervical cancer in a hypoxic tumour-associated macrophage-dependent manner

doi: 10.1007/s10456-020-09766-2

Figure Lengend Snippet: IL-10 derived from hypoxic TAMs is required to maintain LVEM. a The different cytokines expression profiles among M0-N, M0-H, TAM-N and TAM-H were analysed by cytokine array (RayBio GSM-CAA-4000). b Screening and analysis of the differentially expressed cytokines. c The expressions of the five significant cytokines were analysed by qRT-PCR. d The secretions of the five significant cytokines were analysed by ELISA. e The migration effects of hypoxic TAMs-treated HDLECs on tumour cells (SiHa) and M2-polarized THP-1 macrophages were analysed by transwell assay in vitro. “Blank” represents the medium group. f Representative micrographs showing the tube formation in vitro (Scale bar, 50 μm). g Representative images showing the tube formation in vivo (Scale bar, 100 μm). h Statistical analysis showing the length of tube formation. Average length of tubes per field were calculated. i – l Popliteal lymphatic metastasis model was established in female C57BL/6 mice by inoculating the footpad with TC-1 cells (5 × 10 6 ). When footpad tumour size reached 50 mm 3 , IL-10 (50 ng/ml) or PBS was then injected into the centre of the tumours ( n = 5/group, repeated twice) for 2 weeks daily. After 2 weeks of induction, primary tumours reached a comparable size of ~ 150 mm 3 , and then footpad tumours and popliteal LNs were collected for study. i Representative images of LYVE-1 + lymphatic vessel (red), CD206 + TAMs (green) and DAPI (blue) fluorescence staining in footpad tumour. j Metastasis-positive LNs were identified by IHC staining for epithelial marker CK7. k Statistical analysis showing the expression of peritumoural LV and LVEM in footpad tumour. l Statistical analysis showing the ratio of LNM. Error bars represent the mean ± SD of three independent experiments. ** P < 0.01

Article Snippet: Human dermal LECs (HDLECs) were purchased from ScienCell and cultured in endothelial cell medium (ScienCell, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum and endothelial growth medium supplements (ScienCell).

Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Migration, Transwell Assay, In Vitro, In Vivo, Injection, Fluorescence, Staining, Immunohistochemistry, Marker

Journal: Angiogenesis

Article Title: A novel lymphatic pattern promotes metastasis of cervical cancer in a hypoxic tumour-associated macrophage-dependent manner

doi: 10.1007/s10456-020-09766-2

Figure Lengend Snippet: CCL1 mediates the migration of TAMs towards LECs in the formation of LVEM. a Multiple related chemokines were screened in IL-10-activated LECs by qRT-PCR. b Levels of CCL1 secreted by IL-10-activated LECs were measured by ELISA. c The migration effects of CCL1 on tumour cells (SiHa) and M2-polarized THP-1 macrophages were significantly decreased by blockade of CCR8 on receptor cells (siRNA or CCR8 antagonist MC148) or adding anti-CCL1 neutralization antibody in IL-10-activated LEC CM. d – f Popliteal lymphatic metastasis model was established in female C57BL/6 mice by inoculating the footpad with TC-1 cells (5 × 10 6 ). When footpad tumour size reached 50 mm 3 , CCL1 (50 ng/ml) or PBS was then injected into the centre of the tumours ( n = 5/group, repeated twice) for 2 weeks daily. After 2 weeks of induction, primary tumours reached a comparable size of ~ 150 mm 3 , and then popliteal LNs were collected for study. d Representative images of LYVE-1 + lymphatic vessel (red), CD206 + TAMs (green) and DAPI (blue) fluorescence staining in footpad tumour. e IHC Staining of CK7 in popliteal LNs from mice treated with CCL1 (Scale bar, 100 μm). f Statistical analysis showing the ratio of LNM. Error bars represent the mean ± SD of three independent experiments. ** P < 0.01. * P < 0.05

Article Snippet: Human dermal LECs (HDLECs) were purchased from ScienCell and cultured in endothelial cell medium (ScienCell, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum and endothelial growth medium supplements (ScienCell).

Techniques: Migration, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Neutralization, Injection, Fluorescence, Staining, Immunohistochemistry

Journal: Angiogenesis

Article Title: A novel lymphatic pattern promotes metastasis of cervical cancer in a hypoxic tumour-associated macrophage-dependent manner

doi: 10.1007/s10456-020-09766-2

Figure Lengend Snippet: Sp1 high LECs are fundamental to LVEM formation and lymphatic metastasis. a Bioinformatic prediction and screening of potential transcription factors of CCL1. b RNA sequence of the predicted Sp1-binding site to CCL1 was TCCCCTCCCCC. A dual-luciferase reporter assay system was performed to demonstrate the direct binding of Sp1 to the CCL1 promoter region. Transient co-transfection of pcDNA3.1-Sp1 construct with CCL1 promoter into both 293T cells and HDLECs resulted in a significant increase in firefly luciferase activity relative to co-transfection of pcDNA3.1-NC construct with CCL1 promoter or the negative control. c EILSA analysis showed that CCL1 upregulation was detected in Sp1-transduced HDLEC CM, and CCL1 was decreased in Sp1-silenced HDLEC CM. d Representative micrographs of the tube formation assay in vitro (upper panel) and in vivo (lower panel) of HDLECs with stable overexpression or silencing of Sp1 (Scale bar, 100 μm). e Statistical analysis showing the length of tube formation. Average length of tubes per field were calculated. f The migration effects of M2-polarized THP-1 macrophages and tumour cells (SiHa) towards CM of HDLECs with stable overexpression or silencing of Sp1 were detected by transwell array in vitro. g Immunofluorescence staining was applied to analyse Sp1 (purple), LYVE-1 (red), CD163 (green) and DAPI (blue) expression in CSCC tissues (Scale bar, 100 μm). h The correlation between Sp1 expression and LVEM density was statistically analysed by Pearson’s coefficient test (red line, r = 0.5846, P = 0.0009; blue line, r = 0.1018, P = 0.2465). i Immunofluorescence staining was applied to analyse Sp1 (purple), CCL1 (red), CD163 (green) and DAPI (blue) expression in CSCC tissues (Scale bar, 50 μm). j The correlation between Sp1 and CCL1 expression was statistically analysed by Pearson’s coefficient test ( r = 0.4126, P < 0.001). Error bars represent the mean ± SD of three independent experiments. *, P < 0.05

Article Snippet: Human dermal LECs (HDLECs) were purchased from ScienCell and cultured in endothelial cell medium (ScienCell, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum and endothelial growth medium supplements (ScienCell).

Techniques: Sequencing, Binding Assay, Luciferase, Reporter Assay, Cotransfection, Construct, Activity Assay, Negative Control, Tube Formation Assay, In Vitro, In Vivo, Over Expression, Migration, Immunofluorescence, Staining, Expressing