title: Angiopoietin-2 mediates thrombin-induced monocyte adhesion and endothelial permeability.
abstract: Essentials Mechanism of thrombin-induced inflammation is not fully understood. Thrombin induced monocyte adhesion and barrier loss require Angiopoietin-2 (Ang-2). Ang-2 mediates vessel leakage and monocyte adhesion through SHP-2/p38MAPK pathway. Calcium dependent SHP2/p38MAPK activation regulates Ang-2 expression through a feedback loop.
  
  
  
  Background Thrombin imparts an inflammatory phenotype to the endothelium by promoting increased monocyte adhesion and vascular permeability. However, the molecular players that govern these events are incompletely understood. Objective The aim of this study was to determine whether Angiopoietin-2 (Ang-2) has a role, if any, in regulating inflammatory signals initiated by thrombin. Methods Assessment of vascular leakage by Miles assay was performed by intra-dermal injection on the foot paw. Surface levels of intercellular adhesion molecule-1 (ICAM-1) were determined by flow cytometry. Overexpression, knockdown and phosphorylation of proteins were determined by Western blotting. Results In time-course experiments, thrombin-stimulated Ang-2 up-regulation, peaked prior to the expression of adhesion molecule ICAM-1 in human umbilical vein-derived endothelial cells (HUVECs). Knockdown of Ang-2 blocked both thrombin-induced monocyte adhesion and ICAM-1 expression. In addition, Ang-2(-/-) mice displayed defective vascular leakage when treated with thrombin. Introducing Ang-2 protein in Ang-2(-/-) mice failed to recover a wild-type phenotype. Mechanistically, Ang-2 appears to regulate the thrombin-activated calcium spike that is required for tyrosine phosphatase SHP2 and p38 MAPK activation. Further, down-regulation of SHP2 attenuated both thrombin-induced Ang-2 expression and monocyte adhesion. Down-regulation of the adaptor protein Gab1, a co-activator of SHP2, as well as overexpression of the Gab1 mutant incapable of interacting with SHP2 (YFGab1), inhibited thrombin-mediated effects, including downstream activation of p38 MAPK, which in turn was required for Ang-2 expression. Conclusions The data establish an essential role of the Gab1/SHP2/p38MAPK signaling pathway and Ang-2 in regulating thrombin-induced monocyte adhesion and vascular leakage.
authors.affiliation: Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences and Bioengineering, Indian Institute of Technology Madras, Chennai, India.
authors.forename: K
authors.initials: K
authors.lastname: Rathnakumar
authors.affiliation: Division of Vascular Oncology and Metastasis, German Cancer Research Center Heidelberg (DKFZ-ZMBH Alliance), Heidelberg, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Savant
authors.affiliation: Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences and Bioengineering, Indian Institute of Technology Madras, Chennai, India.
authors.forename: H
authors.initials: H
authors.lastname: Giri
authors.affiliation: Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences and Bioengineering, Indian Institute of Technology Madras, Chennai, India.
authors.forename: A
authors.initials: A
authors.lastname: Ghosh
authors.affiliation: Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Fisslthaler
authors.affiliation: Institute for Vascular Signalling, Centre for Molecular Medicine, Goethe University, Frankfurt, Germany.
authors.forename: I
authors.initials: I
authors.lastname: Fleming
authors.affiliation: Seethapathy Clinic and Hospital, Chennai, India.
authors.forename: U
authors.initials: U
authors.lastname: Ram
authors.affiliation: Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences and Bioengineering, Indian Institute of Technology Madras, Chennai, India.
authors.forename: A K
authors.initials: AK
authors.lastname: Bera
authors.affiliation: Division of Vascular Oncology and Metastasis, German Cancer Research Center Heidelberg (DKFZ-ZMBH Alliance), Heidelberg, Germany.|Vascular Biology and Tumor Angiogenesis, Medical Faculty Mannheim (CBTM), Heidelberg University, Heidelberg, Germany.
authors.forename: H G
authors.initials: HG
authors.lastname: Augustin
authors.affiliation: Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences and Bioengineering, Indian Institute of Technology Madras, Chennai, India.
authors.forename: M
authors.initials: M
authors.lastname: Dixit
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D042702:Angiopoietin-2 / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000737:chemistry; D002199:Capillary Permeability; D002448:Cell Adhesion; D004727:Endothelium / Q000378:metabolism*; D005434:Flow Cytometry; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007249:Inflammation; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D020935:MAP Kinase Signaling System; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009000:Monocytes / Q000166:cytology*; D009154:Mutation; D010539:Permeability; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D013917:Thrombin / Q000737:chemistry; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D042702:Angiopoietin-2; C098587:GAB1 protein, human; C573420:ICAM1 protein, human; D034741:RNA, Small Interfering; D018799:Intercellular Adhesion Molecule-1; D048051:p38 Mitogen-Activated Protein Kinases; C516724:PTPN11 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D013917:Thrombin; D002118:Calcium
keywords: GAB1 protein; angiopoietin-2; inflammation; protein tyrosine phosphatase, non-receptor type 11; thrombin