Thrombomodulin is essential for maintaining quiescence in vascular endothelial cells
Thrombomodulin (TM) is a thrombin receptor on endothelial cells that is involved in promoting activation of the anticoagulant protein C pathway during blood coagulation. TM also exerts protective anti-inflammatory properties through a poorly understood mechanism. In this study, we investigated the i...
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| Vydané v: | Proceedings of the National Academy of Sciences - PNAS Ročník 118; číslo 11 |
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| Hlavní autori: | , , , , , |
| Médium: | Journal Article |
| Jazyk: | English |
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United States
16.03.2021
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| ISSN: | 1091-6490, 1091-6490 |
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| Abstract | Thrombomodulin (TM) is a thrombin receptor on endothelial cells that is involved in promoting activation of the anticoagulant protein C pathway during blood coagulation. TM also exerts protective anti-inflammatory properties through a poorly understood mechanism. In this study, we investigated the importance of TM signaling to cellular functions by deleting it from endothelial cells by CRISPR-Cas9 technology and analyzed the resultant phenotype of TM-deficient (
) cells. Deficiency of TM in endothelial cells resulted in increased basal permeability and hyperpermeability when stimulated by thrombin and TNF-α. The loss of the basal barrier permeability function was accompanied by increased tyrosine phosphorylation of VE-cadherin and reduced polymerization of F-actin filaments at cellular junctions. A significant increase in basal NF-κB signaling and expression of inflammatory cell adhesion molecules was observed in
cells that resulted in enhanced adhesion of leukocytes to
cells in flow chamber experiments. There was also a marked increase in expression, storage, and release of the von Willebrand factor (VWF) and decreased storage and release of angiopoietin-2 in
cells. In a flow chamber assay, isolated platelets adhered to
cells, forming characteristic VWF-platelet strings. Increased VWF levels and inflammatory foci were also observed in the lungs of tamoxifen-treated ERcre-TM
mice. Reexpression of the TM construct in
cells, but not treatment with soluble TM, normalized the cellular phenotype. Based on these results, we postulate cell-bound TM endows a quiescent cellular phenotype by tightly regulating expression of procoagulant, proinflammatory, and angiogenic molecules in vascular endothelial cells. |
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| AbstractList | Thrombomodulin (TM) is a thrombin receptor on endothelial cells that is involved in promoting activation of the anticoagulant protein C pathway during blood coagulation. TM also exerts protective anti-inflammatory properties through a poorly understood mechanism. In this study, we investigated the importance of TM signaling to cellular functions by deleting it from endothelial cells by CRISPR-Cas9 technology and analyzed the resultant phenotype of TM-deficient (
) cells. Deficiency of TM in endothelial cells resulted in increased basal permeability and hyperpermeability when stimulated by thrombin and TNF-α. The loss of the basal barrier permeability function was accompanied by increased tyrosine phosphorylation of VE-cadherin and reduced polymerization of F-actin filaments at cellular junctions. A significant increase in basal NF-κB signaling and expression of inflammatory cell adhesion molecules was observed in
cells that resulted in enhanced adhesion of leukocytes to
cells in flow chamber experiments. There was also a marked increase in expression, storage, and release of the von Willebrand factor (VWF) and decreased storage and release of angiopoietin-2 in
cells. In a flow chamber assay, isolated platelets adhered to
cells, forming characteristic VWF-platelet strings. Increased VWF levels and inflammatory foci were also observed in the lungs of tamoxifen-treated ERcre-TM
mice. Reexpression of the TM construct in
cells, but not treatment with soluble TM, normalized the cellular phenotype. Based on these results, we postulate cell-bound TM endows a quiescent cellular phenotype by tightly regulating expression of procoagulant, proinflammatory, and angiogenic molecules in vascular endothelial cells. Thrombomodulin (TM) is a thrombin receptor on endothelial cells that is involved in promoting activation of the anticoagulant protein C pathway during blood coagulation. TM also exerts protective anti-inflammatory properties through a poorly understood mechanism. In this study, we investigated the importance of TM signaling to cellular functions by deleting it from endothelial cells by CRISPR-Cas9 technology and analyzed the resultant phenotype of TM-deficient (TM-/- ) cells. Deficiency of TM in endothelial cells resulted in increased basal permeability and hyperpermeability when stimulated by thrombin and TNF-α. The loss of the basal barrier permeability function was accompanied by increased tyrosine phosphorylation of VE-cadherin and reduced polymerization of F-actin filaments at cellular junctions. A significant increase in basal NF-κB signaling and expression of inflammatory cell adhesion molecules was observed in TM-/- cells that resulted in enhanced adhesion of leukocytes to TM-/- cells in flow chamber experiments. There was also a marked increase in expression, storage, and release of the von Willebrand factor (VWF) and decreased storage and release of angiopoietin-2 in TM-/- cells. In a flow chamber assay, isolated platelets adhered to TM-/- cells, forming characteristic VWF-platelet strings. Increased VWF levels and inflammatory foci were also observed in the lungs of tamoxifen-treated ERcre-TMf/f mice. Reexpression of the TM construct in TM-/- cells, but not treatment with soluble TM, normalized the cellular phenotype. Based on these results, we postulate cell-bound TM endows a quiescent cellular phenotype by tightly regulating expression of procoagulant, proinflammatory, and angiogenic molecules in vascular endothelial cells.Thrombomodulin (TM) is a thrombin receptor on endothelial cells that is involved in promoting activation of the anticoagulant protein C pathway during blood coagulation. TM also exerts protective anti-inflammatory properties through a poorly understood mechanism. In this study, we investigated the importance of TM signaling to cellular functions by deleting it from endothelial cells by CRISPR-Cas9 technology and analyzed the resultant phenotype of TM-deficient (TM-/- ) cells. Deficiency of TM in endothelial cells resulted in increased basal permeability and hyperpermeability when stimulated by thrombin and TNF-α. The loss of the basal barrier permeability function was accompanied by increased tyrosine phosphorylation of VE-cadherin and reduced polymerization of F-actin filaments at cellular junctions. A significant increase in basal NF-κB signaling and expression of inflammatory cell adhesion molecules was observed in TM-/- cells that resulted in enhanced adhesion of leukocytes to TM-/- cells in flow chamber experiments. There was also a marked increase in expression, storage, and release of the von Willebrand factor (VWF) and decreased storage and release of angiopoietin-2 in TM-/- cells. In a flow chamber assay, isolated platelets adhered to TM-/- cells, forming characteristic VWF-platelet strings. Increased VWF levels and inflammatory foci were also observed in the lungs of tamoxifen-treated ERcre-TMf/f mice. Reexpression of the TM construct in TM-/- cells, but not treatment with soluble TM, normalized the cellular phenotype. Based on these results, we postulate cell-bound TM endows a quiescent cellular phenotype by tightly regulating expression of procoagulant, proinflammatory, and angiogenic molecules in vascular endothelial cells. |
| Author | Cai, Xiaofeng Weiler, Hartmut Biswas, Indranil Panicker, Sumith R Giri, Hemant Rezaie, Alireza R |
| Author_xml | – sequence: 1 givenname: Hemant surname: Giri fullname: Giri, Hemant organization: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104 – sequence: 2 givenname: Sumith R orcidid: 0000-0001-8550-9436 surname: Panicker fullname: Panicker, Sumith R organization: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104 – sequence: 3 givenname: Xiaofeng surname: Cai fullname: Cai, Xiaofeng organization: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104 – sequence: 4 givenname: Indranil orcidid: 0000-0003-2479-0249 surname: Biswas fullname: Biswas, Indranil organization: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104 – sequence: 5 givenname: Hartmut surname: Weiler fullname: Weiler, Hartmut organization: Blood Research Institute, Blood Center of Wisconsin, Milwaukee, WI 53226 – sequence: 6 givenname: Alireza R orcidid: 0000-0002-5302-0553 surname: Rezaie fullname: Rezaie, Alireza R email: ray-rezaie@omrf.org organization: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104 |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/33836597$$D View this record in MEDLINE/PubMed |
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