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: 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