Journal: The Journal of Biological Chemistry

Article Title: Platelet Activation Receptor CLEC-2 Regulates Blood/Lymphatic Vessel Separation by Inhibiting Proliferation, Migration, and Tube Formation of Lymphatic Endothelial Cells *

doi: 10.1074/jbc.M111.329987

Figure Lengend Snippet: Inhibitory effects of platelets on endothelial cell proliferation. Platelets inhibited hLEC proliferation but not HUVEC proliferation, depending on CLEC-2. A, cell proliferation of hLECs (upper panels) and HUVECs (lower panels) in the presence of buffer (left panels) and hPlt (right panels, 1 × 108/ml) was investigated by thymidine analog incorporation assay. A group of EdU-incorporated cells is indicated by arrows. B and C, cell proliferation of hLECs (B) and HUVECs (C) in the presence of buffer, hPlt (1 × 108/ml), mPlt WT (1 × 108/ml), and CLEC-2-deficient murine washed platelets (mPlt KO, 1 × 108/ml) was investigated by thymidine analog incorporation assay. Quantification of the proliferation was performed as described under “Experimental Procedures.” The graph illustrates percent change ± S.E. from base line (buffer) (n = 10 from four independent experiments).

Article Snippet: Cells Human umbilical vein endothelial cells (HUVECs) and human lymphatic endothelial cells (hLECs) were purchased from Lonza (Basel, Switzerland) and maintained on culture dishes in endothelial growth medium-2 (EGM-2; Lonza) supplemented with 5% FBS and an EGM-2 microvascular set (0.5 ml of human EGF, 0.2 ml of hydrocortone, 25 ml of FBS, 0.5 ml of VEGF, 2 ml of human FGF-B, 0.5 ml of R3-IGF-1, 0.5 ml of ascorbic acid, and 0.5 ml of GA-1000).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Platelet Activation Receptor CLEC-2 Regulates Blood/Lymphatic Vessel Separation by Inhibiting Proliferation, Migration, and Tube Formation of Lymphatic Endothelial Cells *

doi: 10.1074/jbc.M111.329987

Figure Lengend Snippet: Inhibitory effects of platelets on tube formation of endothelial cells through CLEC-2. A and B, tube formation of hLECs (4 × 105/ml) (A) or HUVECs (5 × 105/ml) (B) in the presence (hPlt) or absence (buffer) of human washed platelets (1 × 108/ml). Images are representative of five different experiments. C, quantification of tube formation. D, tube formation of mLECs (1.25 × 105/ml) in the presence of buffer, wild-type mPlt WT (1 × 107/ml), and CLEC-2-deficient murine washed platelets (mPlt KO, 1 × 107/ml). Images are representative of three different experiments. E, quantification of mLEC tube formation. The graphs in C and E show quantification of tube formation as percent change ± S.E. from base line (buffer) (n = 10–12 from three independent experiments). Three asterisks denote p < 0.005.

Article Snippet: Cells Human umbilical vein endothelial cells (HUVECs) and human lymphatic endothelial cells (hLECs) were purchased from Lonza (Basel, Switzerland) and maintained on culture dishes in endothelial growth medium-2 (EGM-2; Lonza) supplemented with 5% FBS and an EGM-2 microvascular set (0.5 ml of human EGF, 0.2 ml of hydrocortone, 25 ml of FBS, 0.5 ml of VEGF, 2 ml of human FGF-B, 0.5 ml of R3-IGF-1, 0.5 ml of ascorbic acid, and 0.5 ml of GA-1000).

Techniques:

Journal: Oncology Letters

Article Title: Culture medium of bone marrow-derived human mesenchymal stem cells effects lymphatic endothelial cells and tumor lymph vessel formation

doi: 10.3892/ol.2015.2868

Figure Lengend Snippet: Effects of human bone marrow mesenchymal stem cell culture medium (hBM-MSC-CM) on tube formation ability. hBM-MSC-CM enhanced the tube formation ability of lymphatic endothelial cells (LECs). (A) LECs (1×10 4 /well) were incubated with either (a) Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) or (b) one-half DMEM supplemented with 10% FBS and one-half hBM-MSC-CM in the upper Matrigel chamber. Images of the tube-like structures were captured using a microscope (scale bar, 50μm). (c) Statistical analysis was performed using a t-test. ** P<0.01 compared with the DMEM group. (B) (a)Tube formation ability of HGC-27 cells, (b) hBM-MSC-CM-treated HGC-27 cells and (c) LECs.

Article Snippet: The primary lymphatic endothelial cells (LECs) were purchased from ScienCell (Carlsbad, CA, USA).

Techniques: Stem Cell Culture, Incubation, Modification, Microscopy

Journal: Oncology Letters

Article Title: Culture medium of bone marrow-derived human mesenchymal stem cells effects lymphatic endothelial cells and tumor lymph vessel formation

doi: 10.3892/ol.2015.2868

Figure Lengend Snippet: Transwell migration assay. (A) Human bone marrow mesenchymal stem cell culture medium (hBM-MSC-CM) increased the migration ability of lymphatic endothelial cells (LECs) in the (a) control and (b) 50% hBM-MSC-CM-pretreated groups (magnification, ×100). (B) The number of migrated cells in the groups was analyzed using a t-test. * P<0.05 compared with the control group.

Article Snippet: The primary lymphatic endothelial cells (LECs) were purchased from ScienCell (Carlsbad, CA, USA).

Techniques: Transwell Migration Assay, Stem Cell Culture, Migration, Control

Journal: Oncology Letters

Article Title: Culture medium of bone marrow-derived human mesenchymal stem cells effects lymphatic endothelial cells and tumor lymph vessel formation

doi: 10.3892/ol.2015.2868

Figure Lengend Snippet: Western blot analysis revealing the effect of human bone marrow mesenchymal stem cell culture medium (hBM-MSC-CM) on lymphatic endothelial cell (LEC)-specific markers. (A) Expression of vascular epidermal growth factor receptor-3 (VEGFR-3), Prox-1, lymphatic vessel endothelial receptor-1 (LYVE-1) and podoplanin in LECs incubated for 48 h with either Dulbecco’s modified Eagle’s medium (DMEM) or hBM-MSC-CM. (B) Expression of Prox-1 in LECs and GFP-MCF-7 and SGC-7901 cells incubated with either DMEM or hBM-MSC-CM for 40 days, and the expression of VEGFR-3 in HGC-27 cells incubated with either DMEM or hBM-MSC-CM for five and 10 days.

Article Snippet: The primary lymphatic endothelial cells (LECs) were purchased from ScienCell (Carlsbad, CA, USA).

Techniques: Western Blot, Stem Cell Culture, Expressing, Incubation, Modification

Journal: Oncology Letters

Article Title: Culture medium of bone marrow-derived human mesenchymal stem cells effects lymphatic endothelial cells and tumor lymph vessel formation

doi: 10.3892/ol.2015.2868

Figure Lengend Snippet: MTT assay analysis revealing the proliferation of lymphatic endothelial cells (LECs) treated with various concentrations of human bone marrow mesynchymal stem cell culture medium (hBM-MSC-CM) and Dulbecco’s modified Eagle’s medium (DMEM). The concentration of hBM-MSC-CM did not effect the proliferation of primary LECs compared with the control. hBM, human bone marrow; OD, optical density.

Article Snippet: The primary lymphatic endothelial cells (LECs) were purchased from ScienCell (Carlsbad, CA, USA).

Techniques: MTT Assay, Stem Cell Culture, Modification, Concentration Assay, Control

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: Journal of hepatology

Article Title: Platelet-Derived Growth Factor-D Enables Liver Myofibroblasts to Promote Tumor Lymphangiogenesis in Cholangiocarcinoma

doi: 10.1016/j.jhep.2018.12.004

Figure Lengend Snippet: (A-B) In human archival paraffin sections, LMVD was more extensively represented in CCA compared to HCC, as shown by IHC for podoplanin and Lyve-1 (lymphatic endothelial cell marker). (C) On the contrary, BMVD, evaluated as number of CD34+ (blood endothelial cell marker) cells, was increased in HCC samples. Right-side the plots, representative pictures of podoplanin+ (A), Lyve-1+ (B), and CD34+ (C) structures are shown for CCA and HCC; some faint expression of podoplanin is expressed also by CAF. n=6; *p<0.01, using two-tail t test. Original magnification: 200x.

Article Snippet: Cell lines Human lymphatic endothelial cells (LEC, purchased from ScienCell TM ) and human male EGI-1 cells (PDGF-D expressing extrahepatic CCA cell line, purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen) were employed for in vitro experiments.

Techniques: Marker, Expressing

Journal: Journal of hepatology

Article Title: Platelet-Derived Growth Factor-D Enables Liver Myofibroblasts to Promote Tumor Lymphangiogenesis in Cholangiocarcinoma

doi: 10.1016/j.jhep.2018.12.004

Figure Lengend Snippet: (A) In Fischer 344 male rats transplanted with BDE-neu rat CCA cells, selective depletion of CAF by navitoclax was accompanied by a significant decrease in Lyve-1+ LEC without affecting CD31+ blood endothelial cells compared to untreated rats. Up-sided, representative images of CCA sections, with dual immunofluorescence for CD31 (red) and Lyve-1 (green), show the stark differences in lymphatic and blood vessels between navitoclax and vehicle groups. (B) Concomitantly, navitoclax led to a reduction in the number of lymph node metastases that was significant at the peritoneal region (p<0.05), and close to significance at the paraortic region (p=0.068). (n=6 for each group). Original magnification: 100x. **p<0.01 vs Vehicle, using two-tail t test.

Article Snippet: Cell lines Human lymphatic endothelial cells (LEC, purchased from ScienCell TM ) and human male EGI-1 cells (PDGF-D expressing extrahepatic CCA cell line, purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen) were employed for in vitro experiments.

Techniques: Immunofluorescence