title: Heparin-integrin interaction in endothelial cells: downstream signaling and heparan sulfate expression.
abstract: Endothelial cells (ECs) are a source of physiologically important molecules that are synthesized and released to the blood and/or to the subendothelial extracellular matrix such as a heparan sulfate proteoglycan (HSPG) with antithrombotic properties. Previously, we have shown that heparin stimulates the synthesis and modifies the sulfation pattern of this HSPG. Here the molecular mechanisms involved in the up-regulation of HSPG synthesis by heparin in endothelial cells were decoded. The cells were stimulated with heparin and the expression of HSPG and intracellular pathways were evaluated by a combination of methods involving confocal microscopy, flow cytometry, Western blotting analyses, and [(35) S]-sulfate metabolically labeling of the cells. We observed that the up-regulation of HSPG synthesis evoked by heparin is dependent on the interaction of heparin with integrin since RGD peptide abolishes the effect. The activation of integrin leads to tyrosine-phosphorylation of focal adhesion-associated proteins such as FAK, Src, and paxillin. In addition, heparin induces ERK1/2 phosphorylation and inhibitors of Ras and MEK decreased heparin-dependent HSPG synthesis. Moreover, heparin also induced intracellular Ca(2+) release, PLCγ1 (phospholipase Cγ1) and CaMKII (calcium calmodulin kinase II) activation, as well as an increase in nitric oxide (NO) production. Finally, an intracellular Ca(2+) chelator, Ca(2+) signaling inhibitors, and an endothelial NO synthase inhibitor were all able to abolish the effect in heparan sulfate synthesis. In conclusion, the heparin-induced up-regulation of HSPG expression is associated with the phosphorylation of focal adhesion proteins and Ras/Raf/MEK/ERK MAP and Ca(2+) /NO pathways.
authors: Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors: Valquíria P
authors: VP
authors: Medeiros
authors: Edgar J
authors: EJ
authors: Paredes-Gamero
authors: Hugo P
authors: HP
authors: Monteiro
authors: Hugo A O
authors: HA
authors: Rocha
authors: Edvaldo S
authors: ES
authors: Trindade
authors: Helena B
authors: HB
authors: Nader
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000378:metabolism / Q000494:pharmacology; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D002448:Cell Adhesion; D042783:Endothelial Cells / Q000187:drug effects*; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000502:physiology*; D006493:Heparin / Q000378:metabolism* / Q000494:pharmacology; D006497:Heparan Sulfate / Q000235:genetics / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism*; D018613:Microscopy, Confocal; D009569:Nitric Oxide; D010766:Phosphorylation; D011485:Protein Binding; D011817:Rabbits; D015854:Up-Regulation
chemical_list: D000925:Anticoagulants; D002118:Calcium; D005353:Fibronectins; D006493:Heparin; D006497:Heparan Sulfate; D016023:Integrins; D009569:Nitric Oxide