title: Endothelium-derived nitric oxide (NO) activates the NO-epidermal growth factor receptor-mediated signaling pathway in bradykinin-stimulated angiogenesis.
abstract: Nitric oxide (NO) is involved in angiogenesis and stimulates the EGF-R signaling pathway. Stimulation of different endothelial cell lines with bradykinin (BK) activates the endothelial NO synthase (eNOS) and promotes EGF-R tyrosine phosphorylation. Increase in NO production correlated with enhanced phosphorylation of tyrosine residues and S-nitrosylation of the EGF-R. NO-mediated stimulatory effects on tyrosine phosphorylation of the EGF-R, where cGMP independent. Inhibition of soluble guanylyl cyclase followed by BK stimulation of human umbilical vein endothelial cells (HUVECs) did not change tyrosine phosphorylation levels of EGF-R. BK-stimulation of HUVEC promoted S-nitrosylation of the phosphatase SHP-1 and of p21Ras. Phosphorylation and activation of the ERK1/2 MAP kinases mediated by BK was dependent on the activation of the B2 receptor, of the EGF-R, and of p21 Ras. Inhibition of BK-stimulated S-nitrosylation prevented the activation of the ERK1/2 MAP kinases. Furthermore, activated ERK1/2 MAP kinases inhibited internalization of EGF-R by phosphorylating specific Thr residues of its cytoplasmic domain. BK-induced proliferation of endothelial cells was partially inhibited by the NOS inhibitor (L-NAME) and by the MEK inhibitor (PD98059). BK stimulated the expression of vascular endothelial growth factor (VEGF). VEGF expression was dependent on the activation of the EGF-R, the B2 receptor, p21Ras, and on NO generation. A Matrigel®-based in vitro assay for angiogenesis showed that BK induced the formation of capillary-like structures in HUVEC, but not in those cells expressing a mutant of the EGF-R lacking tyrosine kinase activity. Additionally, pre-treatment of BK-stimulated HUVEC with L-NAME, PD98059, and with SU5416, a specific inhibitor of VEGFR resulted in inhibition of in vitro angiogenesis. Our findings indicate that BK-mediated angiogenesis in endothelial cells involves the induction of the expression of VEGF associated with the activation of the NO/EGF-R/p21Ras/ERK1/2 MAP kinases signaling pathway.
authors: Department of Biochemistry, Instituto de Química - Universidade de São Paulo, São Paulo, Brazil.
authors: Miriam S
authors: MS
authors: Moraes
authors: Department of Biochemistry, Center for Cellular and Molecular Therapy - CTCMol, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors: Paulo E
authors: PE
authors: Costa
authors: Department of Microbiology, Immunology, and Parasitology, Universidade Federal de São Paulo, São Paulo, SP, Brazil; Department of Biological Sciences, Universidade Federal de São Paulo/Campus Diadema, SP, Brazil.
authors: Wagner L
authors: WL
authors: Batista
authors: Department of Microbiology, Immunology, and Parasitology, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors: Taysa
authors: T
authors: Paschoalin
authors: Department of Biochemistry, Center for Cellular and Molecular Therapy - CTCMol, Universidade Federal de São Paulo, São Paulo, SP, Brazil; Department of Microbiology, Immunology, and Parasitology, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors: Marli F
authors: MF
authors: Curcio
authors: Department of Biochemistry, Center for Cellular and Molecular Therapy - CTCMol, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors: Roberta E
authors: RE
authors: Borges
authors: Department of Surgery, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
authors: Murched O
authors: MO
authors: Taha
authors: Department of Medicine, Institute for Transformative Molecular Medicine, Case Western University, Cleveland, OH, USA.
authors: Fábio V
authors: FV
authors: Fonseca
authors: Department of Pharmacology, New York University School of Medicine, New York, NY, USA; School of Medicine, Universidad Spíritu Santo - Ecuador, Guayaquil, Ecuador.
authors: Arnold
authors: A
authors: Stern
authors: Department of Biochemistry, Center for Cellular and Molecular Therapy - CTCMol, Universidade Federal de São Paulo, São Paulo, SP, Brazil. Electronic address: hpmonte@uol.com.br.
authors: Hugo P
authors: HP
authors: Monteiro
mesh_terms: D043925:Angiogenesis Inducing Agents / Q000494:pharmacology*; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000378:metabolism / Q000473:pathology; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D009569:Nitric Oxide / Q000096:biosynthesis / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D011817:Rabbits; D026403:S-Nitrosothiols / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D014443:Tyrosine / Q000378:metabolism
chemical_list: D043925:Angiogenesis Inducing Agents; D026403:S-Nitrosothiols; D009569:Nitric Oxide; D014443:Tyrosine; D066246:ErbB Receptors; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D001920:Bradykinin
keywords: Angiogenesis; Bradykinin; Endothelial cells; Epidermal growth factor receptor; Nitric oxide; S-nitrosylation