Podoplanin-Fc reduces lymphatic vessel formation in vitro and in vivo and causes disseminated intravascular coagulation when transgenically expressed in the skin

Abstract Podoplanin is a small transmembrane protein required for development and function of the lymphatic vascular system. To investigate the effects of interfering with its function, we produced an Fc fusion protein of its ectodomain. We found that podoplanin-Fc inhibited several functions of cultured lymphatic endothelial cells and also specifically suppressed lymphatic vessel growth, but not blood vessel growth, in mouse embryoid bodies in vitro and in mouse corneas in vivo. Using a keratin 14 expression cassette, we created transgenic mice that overexpressed podoplanin-Fc in the skin. No obvious outward phenotype was identified in these mice, but surprisingly, podoplanin-Fc—although produced specifically in the skin—entered the blood circulation and induced disseminated intravascular coagulation, characterized by microthrombi in most organs and by thrombocytopenia, occasionally leading to fatal hemorrhage. These findings reveal an important role of podoplanin in lymphatic vessel formation and indicate the potential of podoplanin-Fc as an inhibitor of lymphangiogenesis. These results also demonstrate the ability of podoplanin to induce platelet aggregation in vivo, which likely represents a major function of lymphatic endothelium. Finally, keratin 14 podoplanin-Fc mice represent a novel genetic animal model of disseminated intravascular coagulation.

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Complement Independent Lipopolysaccharide (LPS) Induced Hypotension and Disseminated Intravascular Coagulation: a Correlation of LPS Structure with in Vivo and in Vitro Biological Activities

Advances in Experimental Medicine and Biology - The Reticuloendothelial System in Health and Disease ◽

10.1007/978-1-4684-3297-8_28 ◽

1976 ◽

pp. 339-349 ◽

Cited By ~ 1

Author(s):

R. J. Ulevitch ◽

D. C. Morrison ◽

C. G. Cochrane ◽

P. M. Henson

Keyword(s):

Disseminated Intravascular Coagulation ◽

Biological Activities ◽

Induced Hypotension ◽

Intravascular Coagulation

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Matrix metalloproteinase-2 governs lymphatic vessel formation as an interstitial collagenase

Blood ◽

10.1182/blood-2011-12-400267 ◽

2012 ◽

Vol 119 (21) ◽

pp. 5048-5056 ◽

Cited By ~ 54

Author(s):

Benoit Detry ◽

Charlotte Erpicum ◽

Jenny Paupert ◽

Silvia Blacher ◽

Catherine Maillard ◽

...

Keyword(s):

Matrix Metalloproteinase ◽

Lymphatic Vessel ◽

Type I Collagen ◽

3D Culture ◽

Matrix Composition ◽

Matrix Metalloproteinase 2 ◽

Type I ◽

Vessel Formation

Abstract Lymphatic dysfunctions are associated with several human diseases, including lymphedema and metastatic spread of cancer. Although it is well recognized that lymphatic capillaries attach directly to interstitial matrix mainly composed of fibrillar type I collagen, the interactions occurring between lymphatics and their surrounding matrix have been overlooked. In this study, we demonstrate how matrix metalloproteinase (MMP)–2 drives lymphatic morphogenesis through Mmp2-gene ablation in mice, mmp2 knockdown in zebrafish and in 3D-culture systems, and through MMP2 inhibition. In all models used in vivo (3 murine models and thoracic duct development in zebrafish) and in vitro (lymphatic ring and spheroid assays), MMP2 blockage or down-regulation leads to reduced lymphangiogenesis or altered vessel branching. Our data show that lymphatic endothelial cell (LEC) migration through collagen fibers is affected by physical matrix constraints (matrix composition, density, and cross-linking). Transmission electron microscopy and confocal reflection microscopy using DQ-collagen highlight the contribution of MMP2 to mesenchymal-like migration of LECs associated with collagen fiber remodeling. Our findings provide new mechanistic insight into how LECs negotiate an interstitial type I collagen barrier and reveal an unexpected MMP2-driven collagenolytic pathway for lymphatic vessel formation and morphogenesis.

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Thrombin Does Not Alter Vascular Hyporeactivity in Models of Endotoxin-Induced Septic Shock in Rats

Clinical Science ◽

10.1042/cs0880149 ◽

1995 ◽

Vol 88 (2) ◽

pp. 149-157 ◽

Cited By ~ 1

Author(s):

Viviane Martin ◽

Marie-Louise Wiesel ◽

Anne Albert ◽

Alain Beretz

Keyword(s):

Nitric Oxide ◽

Disseminated Intravascular Coagulation ◽

Vascular Tone ◽

Endotoxic Shock ◽

Vascular Wall ◽

Anticoagulant Treatment ◽

Intravascular Coagulation ◽

Vascular Hyporeactivity

1. Hypotension and vascular hyporesponsiveness to vasoconstrictors are observed during endotoxic shock, and are associated with increased production of nitric oxide in the vascular wall. Disseminated intravascular coagulation is another feature of septicaemia. We hypothesized that thrombin generated during disseminated intravascular coagulation might modulate the changes in vascular tone induced by endotoxin. 2. Incubation of rat aortic rings for 4 h with α-thrombin (0.003–3.0 NIH units/ml) did not change their reactivity to noradrenaline. Incubation for 4 h with lipopolysaccharide increased the EC50 for noradrenaline, whereas co-incubation of thrombin (0.5 NIH units/ml) with lipopolysaccharide did not alter this hyporeactivity to noradrenaline. 3. In vivo in rats, lipopolysaccharide caused early (1 h) and late (4–6 h) hyporeactivity to noradrenaline. In rats infused with lipopolysaccharide and heparin (1 U min−1 kg−1, 0.4 ml/h) or hirudin (2.2 mg ml−1 kg−1, 0.8 ml/h), vasopressor responses to noradrenaline were not different from those after infusion of lipopolysaccharide alone. Aortic rings taken from rats receiving both anticoagulant treatment and lipopolysaccharide had the same sensitivity to noradrenaline as those obtained from rats receiving lipopolysaccharide alone. 4. Our results suggest that, in vivo, disseminated intravascular coagulation does not modify the early and late effects of lipopolysaccharide on arterial pressure and that, in vitro, thrombin neither induces hyporeactivity to noradrenaline nor modifies lipopolysaccharide-induced hyporeactivity. We propose that thrombin generated during disseminated intravascular coagulation in rats does not play a major role in the alterations of vascular tone observed during endotoxic shock.

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A Novel Platelet Activating Factor Antagonist, SM-12502, Attenuates Endotoxin-induced Disseminated Intravascular Coagulation and Acute Pulmonary Vascular Injury by Inhibiting TNF Production in Rats

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650402 ◽

1996 ◽

Vol 75 (06) ◽

pp. 965-970 ◽

Cited By ~ 10

Author(s):

Kazunori Murakami ◽

Kenji Okajima ◽

Mitsuhiro Uchiba ◽

Masayoshi Johno ◽

Hiroaki Okabe ◽

...

Keyword(s):

Disseminated Intravascular Coagulation ◽

Degradation Products ◽

Platelet Activating Factor ◽

Pulmonary Vascular Permeability ◽

Intravascular Coagulation ◽

Tnf Α ◽

Histologic Changes ◽

Factor Antagonist

SummaryAdult respiratory distress syndrome and disseminated intravascular coagulation are important pathologic conditions affecting the outcome of patients with sepsis. To elucidate the possible therapeutic efficacy of SM-12502, a novel platelet activating factor antagonist, on acute lung injury and disseminated intravascular coagulation in sepsis, we investigated the effect of SM-12502 on an endotoxin (ET)-induced septic model in rats. SM-12502 prevented ET-induced increases in pulmonary vascular permeability and ET-induced histologic changes, such as leukocyte infiltration and pulmonary interstitial edema, 6 h following the administration of ET (5 mg/kg). SM-12502 also inhibited the decrease in fibrinogen and the increase in fibrin and fibrinogen degradation products observed following ET administration. SM-12502 prevented increases in the serum concentration of tumor necrosis factor (TNF) 90 min following ET administration in vivo, and significantly inhibited the production of TNF-α by ET-stimulated monocytes in vitro.These findings suggest that SM-12502 attenuates the actions of endotoxin by the inhibition of TNF production

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Sphingosine-1-phosphate promotes lymphangiogenesis by stimulating S1P1/Gi/PLC/Ca2+ signaling pathways

Blood ◽

10.1182/blood-2007-11-125203 ◽

2008 ◽

Vol 112 (4) ◽

pp. 1129-1138 ◽

Cited By ~ 68

Author(s):

Chang Min Yoon ◽

Bok Sil Hong ◽

Hyung Geun Moon ◽

Seyoung Lim ◽

Pann-Ghill Suh ◽

...

Keyword(s):

Signaling Pathways ◽

Lymphatic Vessel ◽

Molecular Mechanisms ◽

Inflammatory Responses ◽

Lymphatic Vessels ◽

Tissue Fluid ◽

Vessel Formation ◽

Sphingosine 1 Phosphate

Abstract The lymphatic system plays pivotal roles in mediating tissue fluid homeostasis and immunity, and excessive lymphatic vessel formation is implicated in many pathological conditions, which include inflammation and tumor metastasis. However, the molecular mechanisms that regulate lymphatic vessel formation remain poorly characterized. Sphingosine-1-phosphate (S1P) is a potent bioactive lipid that is implicated in a variety of biologic processes such as inflammatory responses and angiogenesis. Here, we first report that S1P acts as a lymphangiogenic mediator. S1P induced migration, capillary-like tube formation, and intracellular Ca2+ mobilization, but not proliferation, in human lymphatic endothelial cells (HLECs) in vitro. Moreover, a Matrigel plug assay demonstrated that S1P promoted the outgrowth of new lymphatic vessels in vivo. HLECs expressed S1P1 and S1P3, and both RNA interference–mediated down-regulation of S1P1 and an S1P1 antagonist significantly blocked S1P-mediated lymphangiogenesis. Furthermore, pertussis toxin, U73122, and BAPTA-AM efficiently blocked S1P-induced in vitro lymphangiogenesis and intracellular Ca2+ mobilization of HLECs, indicating that S1P promotes lymphangiogenesis by stimulating S1P1/Gi/phospholipase C/Ca2+ signaling pathways. Our results suggest that S1P is the first lymphangiogenic bioactive lipid to be identified, and that S1P and its receptors might serve as new therapeutic targets against inflammatory diseases and lymphatic metastasis in tumors.

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Angiopoietin-1 promotes LYVE-1-positive lymphatic vessel formation

Blood ◽

10.1182/blood-2004-08-3382 ◽

2005 ◽

Vol 105 (12) ◽

pp. 4649-4656 ◽

Cited By ~ 168

Author(s):

Tohru Morisada ◽

Yuichi Oike ◽

Yoshihiro Yamada ◽

Takashi Urano ◽

Masaki Akao ◽

...

Keyword(s):

Endothelial Cells ◽

Lymphatic Vessel ◽

Matrix Protein ◽

Lymphatic Vessels ◽

Vascular Endothelial ◽

Vessel Formation ◽

Lymphatic Vasculature ◽

In Vitro Effects

Abstract Angiopoietin (Ang) signaling plays a role in angiogenesis and remodeling of blood vessels through the receptor tyrosine kinase Tie2, which is expressed on blood vessel endothelial cells (BECs). Recently it has been shown that Ang-2 is crucial for the formation of lymphatic vasculature and that defects in lymphangiogenesis seen in Ang-2 mutant mice are rescued by Ang-1. These findings suggest important roles for Ang signaling in the lymphatic vessel system; however, Ang function in lymphangiogenesis has not been characterized. In this study, we reveal that lymphatic vascular endothelial hyaluronan receptor 1-positive (LYVE-1+) lymphatic endothelial cells (LECs) express Tie2 in both embryonic and adult settings, indicating that Ang signaling occurs in lymphatic vessels. Therefore, we examined whether Ang-1 acts on in vivo lymphatic angiogenesis and in vitro growth of LECs. A chimeric form of Ang-1, cartilage oligomeric matrix protein (COMP)-Ang-1, promotes in vivo lymphatic angiogenesis in mouse cornea. Moreover, we found that COMP-Ang-1 stimulates in vitro colony formation of LECs. These Ang-1-induced in vivo and in vitro effects on LECs were suppressed by soluble Tie2-Fc fusion protein, which acts as an inhibitor by sequestering Ang-1. On the basis of these observations, we propose that Ang signaling regulates lymphatic vessel formation through Tie2. (Blood. 2005;105:4649-4656)

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Digging deeper into lymphatic vessel formation in vitro and in vivo

BMC Cell Biology ◽

10.1186/1471-2121-12-29 ◽

2011 ◽

Vol 12 (1) ◽

pp. 29 ◽

Cited By ~ 20

Author(s):

Benoit Detry ◽

Françoise Bruyère ◽

Charlotte Erpicum ◽

Jenny Paupert ◽

Françoise Lamaye ◽

...

Keyword(s):

Lymphatic Vessel ◽

Vessel Formation

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The Glycosaminoglycan of Recombinant Human Soluble Thrombomodulin Affects Antithrombotic Activity in a Rat Model of Tissue Factor-Induced Disseminated Intravascular Coagulation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648448 ◽

1992 ◽

Vol 67 (03) ◽

pp. 366-370 ◽

Cited By ~ 16

Author(s):

Katsuhiko Nawa ◽

Teru Itani ◽

Mayumi Ono ◽

Katsu-ichi Sakano ◽

Yasumasa Marumoto ◽

...

Keyword(s):

Tissue Factor ◽

Disseminated Intravascular Coagulation ◽

Rat Model ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Soluble Thrombomodulin ◽

Intravascular Coagulation ◽

Platelet Counts ◽

Recombinant Human Soluble Thrombomodulin

SummaryPrevious studies on recombinant human soluble thrombomodulin (rsTM) from Chinese hamster ovary cells revealed that rsTM was expressed as two proteins that differed functionally in vitro due to the presence (rsTMp) or absence (rsTMa) of chondroitin-4-sulfate. The current study evaluates the in vivo behavior of rsTM in rats and in a rat model of tissue factor-induced disseminated intravascular coagulation (DIC). rsTMp was more potent than rsTMa for prolongation of the activated partial thromboplastin time (APTT) and their in vivo half-lives determined by ELISA were 20 min for rsTMp and 5.0 h for rsTMa. Injection of a tissue factor suspension (5 mg/kg) resulted in DIC as judged by decreased platelet counts and fibrinogen concentrations, prolonged APTT, and increased fibrin and fibrinogen degradation products (FDP) levels. A bolus injection of either rsTM (0.2 mg/kg) 1 min before induction of DIC essentially neutralized effects on platelets, fibrinogen, and FDP levels, and had only a moderate effect on APTT prolongation. The dose of anticoagulant to inhibit the drop in platelet counts by 50% (ED50) was 0.2 mg/kg rsTMa, 0.07 mg/kg rsTMp, and 7 U/ kg heparin. The effect of increasing concentrations of rsTM and heparin on bleeding times were compared in experiments involving incision of the rat tail. Doubling of the bleeding times occurred at 5 mg/kg rsTMa, 3 mg/kg rsTMp or 90 U/kg heparin. These values represent a 25-fold increase over the ED50 for rsTMa, 43-fold for rsTMp and 13-fold for heparin. These results suggest that rsTMp is a potent anticoagulant to inhibit the platelet reduction when injected prior to the induction of DIC in rats.

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Abstract 558: Microchanneled Polysaccharide Hydrogel Laden With Endothelial Cells and Mesenchymal Stem Cells With VEGF Facilitate Formation of Vascular Structures and Are Effective for Repairing Tissue Ischemia

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.34.suppl_1.558 ◽

2014 ◽

Vol 34 (suppl_1) ◽

Author(s):

Sangho Lee ◽

Min Kyung Lee ◽

Hyunjoon Kong ◽

Young-sup Yoon

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Mesenchymal Stem Cells ◽

Cell Survival ◽

Vascular Structure ◽

Hindlimb Ischemia ◽

Alginate Hydrogel ◽

Vessel Formation

Various hydrogels are used to create vascular structure in vitro or to improve cell engraftment to overcome low cell survival in vivo, a main hurdle for bare cell therapy Recently we developed a modified alginate hydrogel within which microchannels are aligned to guide the direction and spatial organization of loaded cells. We investigated whether these cell constructs in which HUVECs and human mesenchymal stem cells (hMSCs) are co-loaded in this novel microchanneled hydrogel facilitate formation of vessels in vitro and in vivo, and enhance recovery of hindlimb ischemia. We crafted a modified alginate hydrogel which has microchannels, incorporates a cell adhesion peptide RGD, and was encapsulated with VEGF. We then compared vascular structure formation between the HUVEC only (2 x 105 cells) group and the HUVEC plus hMSC group. In the HUVEC+hMSC group, we mixed HUVECs and hMSCs at the ratio of 3:1. For cell tracking, we labeled HUVECs with DiO, a green fluorescence dye. After loading cells into the microchannels of the hydrogel, these constructs were cultured for seven days and were examined by confocal microscopy. In the HUVEC only group, HUVECs stands as round shaped cells without forming tubular structures within the hydrogel. However, in the HUVEC+hMSC group, HUVECs were stretched out and connected with each other, and formed vessel-like structure following pre-designed microchannels. These results suggested that hMSCs play a critical role for vessel formation by HUVECs. We next determined their in vivo effects using a mouse hindlimb ischemia model. We found that engineered HUVEC+hMSC group showed significantly higher perfusion over 4 weeks compared to the engineered HUVEC only group or bare cell (HUVEC) group. Confocal microscopic analysis of harvested tissues showed more robust vessel formation within and outside of the cell constructs and longer term cell survival in HUVEC+hMSC group compared to the other groups. In conclusion, this novel microchanneled alginate hydrogel facilitates aligned vessel formation of endothelial cells when combined with MSCs. This vessel-embedded hydrogel constructs consisting of HUVECs and MSCs contribute to perfusable vessel formation, prolong cell survival in vivo, and are effective for recovering limb ischemia.

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