title: Expression of nitric oxide synthase isozymes (NOS I-III) by immunohistochemistry and DNA in situ hybridization. Correlation with macrophage presence, vascular endothelial growth factor (VEGF) and oedema volumetric data in 220 glioblastomas.
abstract: BACKGROUND
  Nitric oxide (NO) is synthesized from arginine by three different isozymes of nitric oxide synthase (NOS I-III). NO has been identified as a powerful metabolite of vascular smooth muscle cell function, cerebral blood circulation and oedema induction. NOS induction by different cytokines has been shown previously in glioblastoma cell cultures and NOS III expression due to astrocytoma grading has been shown in several tumors recently. The aim of the present study was to study the coexpression of NOS I-III, macrophage and capillary presence with VEGF, EGF and their receptors and to investigate a possible mechanism in peritumoral oedema generation.
  
  
  METHODS
  We have investigated the expression (4-grade values, blinded assay by two observers) of NOS I-III together with those of VEGF, VEGF- R (Flt-1), EGF-R1, von-Willebrand-factor (VWF) and a pan-macrophage marker (Ki-M1P) immunohistochemically in tumor specimens from 220 patients and performed tumor volume morphometry by image analysis in a subgroup of 32 cases to test for any correlation with the peritumoral oedema volumes. Inducible NOS II was further investigated by in situ labelling with a DNA oligonucleotide probe cocktail.
  
  
  RESULTS
  All of the specimens revealed some NOS expression, NOS II was expressed in macrophages, microglia and endothelial cells, NOS III and I was localized in glioblastoma cells, NOS III in endothelial cells as well. The highest degrees of expression were observed in 46% (NOS I), 22% (NOS II) and 75% (NOS III) of all specimens. Inducible NOS II in any expression grade was observed in 47.5% of the specimens. Significant correlations were observed for the expression of the macrophage marker Ki-M1P with NOS II (p = 0.024), endothelial NOS III with NOS I (p = 0.0003), VEGF-R1 with NOS II (p = 0.0008) and NOS III (p = 0.011) The oedema volumes could not be correlated significantly with NOS or VEGF-R1 expression values but with those of endothelial staining (p = 0.02). We observed a trend towards higher Ki-M1P expression values together with higher oedema volume extensions. In situ hybridization demonstrated reaction products in endothelial and perivascular regions and sometimes scattered throughout the specimens revealing the labelling of macrophages.
  
  
  CONCLUSIONS
  The main source of NO is NOS I and NOS III. The latter is located in endothelial cells and glioblastoma cells. The expression of NOS II in glioblastomas is restricted to infiltrating macrophages. NOS II and III expressions were observed significantly together with that of VEGF-R1. Neither NOS I-III nor VEGF-R expression could be correlated with the extension of the peritumoral oedema.
authors.affiliation: Georg-August-Universität, Göttingen, Germany. hludwig@gwdg.de
authors.forename: H C
authors.initials: HC
authors.lastname: Ludwig
authors.forename: I
authors.initials: I
authors.lastname: Feiz-Erfan
authors.forename: V
authors.initials: V
authors.lastname: Bockermann
authors.forename: J
authors.initials: J
authors.lastname: Behnke-Mursch
authors.forename: K
authors.initials: K
authors.lastname: Schallock
authors.forename: E
authors.initials: E
authors.lastname: Markakis
mesh_terms: D000911:Antibodies, Monoclonal / Q000032:analysis; D001324:Autoantigens; D001929:Brain Edema / Q000188:drug therapy / Q000209:etiology / Q000473:pathology*; D001932:Brain Neoplasms / Q000098:blood supply / Q000150:complications / Q000201:enzymology* / Q000473:pathology / Q000628:therapy; D003907:Dexamethasone / Q000627:therapeutic use; D016228:Endothelial Growth Factors / Q000032:analysis*; D004790:Enzyme Induction; D004815:Epidermal Growth Factor / Q000032:analysis; D066246:ErbB Receptors / Q000032:analysis; D005260:Female; D005909:Glioblastoma / Q000098:blood supply / Q000150:complications / Q000201:enzymology* / Q000473:pathology / Q000628:therapy; D006801:Humans; D017403:In Situ Hybridization; D007527:Isoenzymes / Q000032:analysis* / Q000096:biosynthesis; D008222:Lymphokines / Q000032:analysis*; D008264:Macrophages / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000032:analysis* / Q000096:biosynthesis; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000096:biosynthesis; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000032:analysis* / Q000096:biosynthesis; D009687:Nuclear Proteins; D046988:Proteasome Endopeptidase Complex; D020794:Receptor Protein-Tyrosine Kinases / Q000032:analysis; D017978:Receptors, Growth Factor / Q000032:analysis; D040262:Receptors, Vascular Endothelial Growth Factor; D015290:Second Messenger Systems; D014057:Tomography, X-Ray Computed; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D014841:von Willebrand Factor / Q000032:analysis
chemical_list: D000911:Antibodies, Monoclonal; D001324:Autoantigens; D016228:Endothelial Growth Factors; D007527:Isoenzymes; C099558:Ki antigen; D008222:Lymphokines; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D017978:Receptors, Growth Factor; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D014841:von Willebrand Factor; D009569:Nitric Oxide; D004815:Epidermal Growth Factor; D003907:Dexamethasone; D019001:Nitric Oxide Synthase; D066246:ErbB Receptors; D020794:Receptor Protein-Tyrosine Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D046988:Proteasome Endopeptidase Complex