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: Bioengineering

Article Title: Fibroblast-Generated Extracellular Matrix Guides Anastomosis during Wound Healing in an Engineered Lymphatic Skin Flap

doi: 10.3390/bioengineering10020149

Figure Lengend Snippet: Timeline of in vitro wound healing model. The culture-insert is first attached to a well plate. The supporting cells, human dermal fibroblasts (HDFs) and mesenchymal stem cells (MSCs), are cultured inside for 9 days in either the MSC-HDF or HDF-HDF configuration. In MSC-HDF, the supporting cells alternate between MSCs and HDFs, whereas HDF-HDF only contain HDFs. Subsequently, lymphatic endothelial cells (LECs) are seeded on top for 10 h of vessel assembly, or lymphangiogenesis, before culture-insert removal. Three inserts were used for each configuration at each imaging timepoint.

Article Snippet: CloneticsTM Dermal Lymphatic Microvascular Endothelial Cells (LECs) and Poietics TM Normal Human Bone Marrow Derived Mesenchymal Stem Cells (MSCs) were obtained from Lonza, Basel, Switzerland.

Techniques: In Vitro, Cell Culture, Imaging

Journal: Bioengineering

Article Title: Fibroblast-Generated Extracellular Matrix Guides Anastomosis during Wound Healing in an Engineered Lymphatic Skin Flap

doi: 10.3390/bioengineering10020149

Figure Lengend Snippet: Engineered lymphatic flap and wound healing model. ( A ) Composition of engineered lymphatic flap. The three layers of the engineered flap (lymphatic capillaries, MSCs, and HDF extracellular matrix (ECM)) mimics the ultra-thin skin flaps used in vascularized lymph vessel transfer (VLVT). ( B ) Mechanism of bilayered wound healing assay. Capillary formation and sprouting is limited by the basal ECM. Supporting cell proliferation and ECM secretion allows lymphangiogenesis over the inhospitable culture plate substrate. ( C ) Immunostaining of engineered flap. LECs (Red). Collagen I (Green). Cell Nuclei (Blue). Scale bar: 100 µm. ( D ) Natural collagen swirls forming in the process of wound healing. Lymphatic capillaries colocalized with these ECM patterns. Wound gaps with detached tissues were excluded for analysis. LECs (red) Collagen I (Green). Wound gap: 500 µm. Scale bar: 13 mm.

Article Snippet: CloneticsTM Dermal Lymphatic Microvascular Endothelial Cells (LECs) and Poietics TM Normal Human Bone Marrow Derived Mesenchymal Stem Cells (MSCs) were obtained from Lonza, Basel, Switzerland.

Techniques: Wound Healing Assay, Immunostaining

Journal: Bioengineering

Article Title: Fibroblast-Generated Extracellular Matrix Guides Anastomosis during Wound Healing in an Engineered Lymphatic Skin Flap

doi: 10.3390/bioengineering10020149

Figure Lengend Snippet: Images of capillary invasion into the wound gap after a 48-h lymphangiogenic period. ( A , B ) Day 4. Supporting cells have closed the wound gap. Some capillaries have formed, but not yet invaded the wound gap. Capillaries were still short and immature. ( C ) LECs fail to form capillaries when seeded directly on the culture dish. Capillary invasion over wound gaps must therefore be over basal cells. ( D – G ) Day 8. Capillaries have invaded the wound gap. MSC-HDF gaps had more connecting capillaries than HDF-HDF. Scale bar: 500 µm.

Article Snippet: CloneticsTM Dermal Lymphatic Microvascular Endothelial Cells (LECs) and Poietics TM Normal Human Bone Marrow Derived Mesenchymal Stem Cells (MSCs) were obtained from Lonza, Basel, Switzerland.

Techniques:

Journal: Bioengineering

Article Title: Fibroblast-Generated Extracellular Matrix Guides Anastomosis during Wound Healing in an Engineered Lymphatic Skin Flap

doi: 10.3390/bioengineering10020149

Figure Lengend Snippet: Fibroblasts guide capillary integration through natural collagen I alignment during in vitro graft integration. ( A ) ECM-mediated anastomosis model, where end-to-end anastomosis of capillaries from opposing wound edges are guided by collagen tracks. ( B – D ) Fibroblast proliferation and migration patterns in the wound gap leave behind collagen tracks that the capillaries follow. Arrows mark the direction of the collagen fibers as well as capillary invasion. LECs (red) Collagen I (Green). Scale bar: 200 µm.

Article Snippet: CloneticsTM Dermal Lymphatic Microvascular Endothelial Cells (LECs) and Poietics TM Normal Human Bone Marrow Derived Mesenchymal Stem Cells (MSCs) were obtained from Lonza, Basel, Switzerland.

Techniques: In Vitro, Migration

Journal: Bioengineering

Article Title: Fibroblast-Generated Extracellular Matrix Guides Anastomosis during Wound Healing in an Engineered Lymphatic Skin Flap

doi: 10.3390/bioengineering10020149

Figure Lengend Snippet: Immunostaining of implanted engineered lymphatic flap. Rat-human chimeric capillaries were found 7 days after subcutaneous implantation in athymic nude rats ( n = 3). Rat, Human PDPN (green), HNA (red). Each row represents a different site of anastomosis. Arrows point to HNA - LECs within HNA + vessels. Scale bar: 50 µm.

Article Snippet: CloneticsTM Dermal Lymphatic Microvascular Endothelial Cells (LECs) and Poietics TM Normal Human Bone Marrow Derived Mesenchymal Stem Cells (MSCs) were obtained from Lonza, Basel, Switzerland.

Techniques: Immunostaining

Journal: eLife

Article Title: Lymphatic endothelium stimulates melanoma metastasis and invasion via MMP14-dependent Notch3 and β1-integrin activation

doi: 10.7554/eLife.32490

Figure Lengend Snippet:

Article Snippet: Adult Dermal lymphatic endothelial cells , , Lonza, Basel, Switzerland , , .

Techniques: Functional Assay, Transfection, Construct, Plasmid Preparation, Sequencing, SYBR Green Assay, Software

Journal: Biomedicines

Article Title: Paneth Cells Regulate Lymphangiogenesis under Control of Microbial Signals during Experimental Portal Hypertension

doi: 10.3390/biomedicines10071503

Figure Lengend Snippet: Lymphangiogenic properties of factors released by PCs in response to PAMPs. ( A ) Representative images showing tube formation assays of LECs co-cultured with CM for 24 h, collected from intestinal organoids unstimulated or stimulated with indicated microbial products in the presence or absence of PCs. Black lines indicate 100 µm magnification. ( B ) Quantification of tube formation assays indicating the number of branched points per field area. ( C ) Representative images showing wound healing assays of LECs co-cultured with CM collected from intestinal organoids unstimulated or stimulated with indicated microbial products in the presence or absence of PCs at time 0 h (T = 0) or after 12 h (T = 12) of incubation. Black lines indicate 50 µm magnification. ( D ) Quantification of wound healing assays indicating the distance (µm) that LECs migrated after 12 h (wound recovery). Abbreviations: CM, conditioned media; CpG, CpG motifs of bacterial DNA; LECs, lymphatic endothelial cells; LPS, lipopolysaccharide; Math1 lox/lox VilCreER T2 , Paneth cell depleted; MDP, muramyl dipeptide; ns, not significant; PGN, peptidoglycan; PCs, Paneth cells. Data are expressed as mean ± SD and are representative of 3 independent experiments. * p < 0.05; ** p < 0.005; *** p < 0.001; **** p < 0.0001.

Article Snippet: The capacity of dermal lymphatic microvascular endothelial cells (LECs) (Cat. No. XSEL6C1, Lonza, Basel, Switzerland) to form lymphatic tubes in vitro was assessed as previously described [ ].

Techniques: Cell Culture, Incubation

Journal: Cell Transplantation

Article Title: Extracellular Vesicles Derived From Hypoxia-Conditioned Adipose-Derived Mesenchymal Stem Cells Enhance Lymphangiogenesis

doi: 10.1177/09636897221107536

Figure Lengend Snippet: Identification of hypoxia ADSC-derived extracellular vesicles. (A) Transmission electron microscopy analysis of the morphology of H-ADSC/evs and N-ADSC/evs. Scale bars: 100 nm. (B) NTA analysis of H-ADSC/exos and N-ADSC/exos. (C) Western blot analysis of CD9, CD63, and TSG-101 expression in of H-ADSC/evs and N-ADSC/evs. (D) Immunofluorescence staining determining the uptake of hypoxia ADSC-derived extracellular vesicles by LECs. Data represent the mean ± SD of three separate experiments; comparison was performed with Student’s t -test. Scale bar: 100 μm. ADSCs: adipose-derived mesenchymal stem cells; H-ADSC/evs: hypoxia ADSC-derived extracellular vesicles; LECs: lymphatic endothelial cells; N-ADSC/evs: normoxia ADSC-derived extracellular vesicles; SD: standard deviation.

Article Snippet: Human ADSCs and dermal lymphatic endothelial cells (LECs) were obtained from Guangzhou Cyagen Biology (Huangpu District, Guangzhou, China).

Techniques: Derivative Assay, Transmission Assay, Electron Microscopy, Western Blot, Expressing, Immunofluorescence, Staining, Comparison, Standard Deviation

Journal: Cell Transplantation

Article Title: Extracellular Vesicles Derived From Hypoxia-Conditioned Adipose-Derived Mesenchymal Stem Cells Enhance Lymphangiogenesis

doi: 10.1177/09636897221107536

Figure Lengend Snippet: Effects of hypoxia ADSC-derived extracellular vesicles on LEC proliferation, migration, and tube formation. (A) CCK-8 assay in LECs after treatment with or without H-ADSC/evs (or N-ADSC/evs). (B) Migration assay in LECs after treatment with or without H-ADSC/evs (or N-ADSC/evs). (C) Tube formation assay in LECs after treatment with or without H-ADSC/evs (or N-ADSC/evs). (D, E) qRT-PCR and Western blot analysis of LYVE-1 and PROX1 expression in LECs after treatment with or without H-ADSC/evs (or N-ADSC/evs). * P < 0.05, ** P < 0.01. Data represent the mean ± SD of three separate experiments; comparison was performed with Student’s t -test. Scale bar: 100 μm. ADSCs: adipose-derived mesenchymal stem cells; CCK-8: Cell Counting Kit-8; H-ADSC/evs: Hypoxia ADSC-derived extracellular vesicles; LECs: lymphatic endothelial cells; N-ADSC/evs: normoxia ADSC-derived extracellular vesicles; mRNA: messenger RNA; SD: standard deviation.

Article Snippet: Human ADSCs and dermal lymphatic endothelial cells (LECs) were obtained from Guangzhou Cyagen Biology (Huangpu District, Guangzhou, China).

Techniques: Derivative Assay, Migration, CCK-8 Assay, Tube Formation Assay, Quantitative RT-PCR, Western Blot, Expressing, Comparison, Cell Counting, Standard Deviation

Journal: Cell Transplantation

Article Title: Extracellular Vesicles Derived From Hypoxia-Conditioned Adipose-Derived Mesenchymal Stem Cells Enhance Lymphangiogenesis

doi: 10.1177/09636897221107536

Figure Lengend Snippet: H-ADSC/evs increase lymphangiogenesis by downregulation of miR-129. (A) qRT-PCR analysis of miRNA expression in LECs treated with H-ADSC/evs or N-ADSC/evs. (B) CCK-8 assay in LECs after treatment with H-ADSC/evs (or N-ADSC/evs) together with miR-129 mimics (or NC mimics). (C) Transwell migration assay in LECs after treatment with H-ADSC/evs (or N-ADSC/evs) together with miR-129 mimics (or NC mimics). (D) Tube formation assay in LECs after treatment with H-ADSC/evs (or N-ADSC/evs) together with miR-129 mimics (or NC mimics). Data represent the mean ± SD of three separate experiments; comparison was performed with Student’s t-test. Scale bar: 100 μm. ADSCs: adipose-derived mesenchymal stem cells; CCK-8: Cell Counting Kit-8; H-ADSC/evs: Hypoxia ADSC-derived extracellular vesicles; LECs: lymphatic endothelial cells; N-ADSC/evs: normoxia ADSC-derived extracellular vesicles; mRNA: messenger RNA; SD: standard deviation. * P < 0.05, ** P < 0.01.

Article Snippet: Human ADSCs and dermal lymphatic endothelial cells (LECs) were obtained from Guangzhou Cyagen Biology (Huangpu District, Guangzhou, China).

Techniques: Quantitative RT-PCR, Expressing, CCK-8 Assay, Transwell Migration Assay, Tube Formation Assay, Comparison, Derivative Assay, Cell Counting, Standard Deviation

Journal: Cell Transplantation

Article Title: Extracellular Vesicles Derived From Hypoxia-Conditioned Adipose-Derived Mesenchymal Stem Cells Enhance Lymphangiogenesis

doi: 10.1177/09636897221107536

Figure Lengend Snippet: Exosomal miR-129 regulates HMGB1 expression. (A) Western blot analysis of HMGB1 expression in LECs after treatment with H-ADSC/evs with miR-129 mimics or NC mimics. (B) The binding site of miR-129 in HMGB1 3’UTR. (C) Luciferase reporter assay in LECs transfected with miR-129 and wild type (wt) or mutated (mut) HMGB1 3’UTR. (D) CCK-8 assay demonstrated that transfection of miR-129 abolished the effect of H-ADSC/evs on LEC viability, and this effect was reversed by overexpression of HMGB1. (E) Transwell migration assay revealed that transfection of miR-129 mimics counteracted the effect of H-ADSC/evs on LEC migration, and this effect was reversed by overexpression of HMGB1. (F) Tube formation assay showed that overexpression of miR-129 restrained the effect of H-ADSC/evs on LEC tube formation, and this effect was reversed by transfection of HMGB1. Data represent the mean ± SD of three separate experiments; comparison was performed with Student’s t-test. Scale bar: 100 μm. ADSCs: adipose-derived mesenchymal stem cells; CCK-8: Cell Counting Kit-8; H-ADSC/evs: Hypoxia ADSC-derived extracellular vesicles; LECs: lymphatic endothelial cells; N-ADSC/evs: normoxia ADSC-derived extracellular vesicles; SD: standard deviation. * P < 0.05, ** P < 0.01.

Article Snippet: Human ADSCs and dermal lymphatic endothelial cells (LECs) were obtained from Guangzhou Cyagen Biology (Huangpu District, Guangzhou, China).

Techniques: Expressing, Western Blot, Binding Assay, Luciferase, Reporter Assay, Transfection, CCK-8 Assay, Over Expression, Transwell Migration Assay, Migration, Tube Formation Assay, Comparison, Derivative Assay, Cell Counting, Standard Deviation

Journal: Cell Transplantation

Article Title: Extracellular Vesicles Derived From Hypoxia-Conditioned Adipose-Derived Mesenchymal Stem Cells Enhance Lymphangiogenesis

doi: 10.1177/09636897221107536

Figure Lengend Snippet: Hypoxia-conditioned ADSC-derived extracellular vesicles activates AKT via HMGB1. (A) Western blot analysis of AKT expression and phosphorylation in LECs after treatment with H-ADSC/evs and transfection with HMGB1 siRNA (si-HMGB1) or control siRNA (si-NC). (B) Treatment with perifosine (30 μM) decreased the tube formation of LECs enhanced by H-ADSC/evs. (D) Treatment with perifosine (30 μM) reduced H-ADSC/evs-induced LYVE-1 and PROX1 expression. Data represent the mean ± SD of three separate experiments; comparison was performed with Student’s t -test. Scale bar: 100 μm. ADSCs: adipose-derived mesenchymal stem cells; H-ADSC/evs: Hypoxia ADSC-derived extracellular vesicles; LECs: lymphatic endothelial cells; N-ADSC/evs: normoxia ADSC-derived extracellular vesicles; SD: standard deviation. * P < 0.05, ** P < 0.01.

Article Snippet: Human ADSCs and dermal lymphatic endothelial cells (LECs) were obtained from Guangzhou Cyagen Biology (Huangpu District, Guangzhou, China).

Techniques: Derivative Assay, Western Blot, Expressing, Phospho-proteomics, Transfection, Control, Comparison, Standard Deviation