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

Background: Nitric oxide (NO) is synthesized from arginine by three differe nt isozymes of nitric oxide synthase (NOS I-III). NO has been identified as a powerful metabolite cf vascular smooth muscle cell function, cerebral bl ood circulation and oedema induction. NOS induction by different cytokines has been shown previously in glioblastoma cell cultures and NOS III express ion due to astrocytoma grading has been shown in several tumors recently. T he aim of the present study was to study the coexpression of NOS I-III, mac rophage and capillary presence with VEGF, ECF and their receptors and to in vestigate a possible mechanism in peritumoral oedema generation.. Materials and 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 -MIP) immunohistochemically in tumor specimens from 220 patients and perfor med tumor volume morphometry by image analysis in a subgroup of 32 cases to test for any correlation with the peritumoral oedema volumes. Inducible NO S II was further investigated by in situ labelling with a DNA oligonucleoti de probe cocktail. Results: All of the specimens revealed some NOS expressi on, NOS II was expressed in macrophages, microglia and endothelial cells, N OS III and I was localized in glioblastoma cells, NOS III in endothelial ce lls 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 correl ations were observed for the expression of the macrophage marker Ki-M1P wit h NOS II (p = 0.024), endothelial NOS III with NOS I (p = 00003), VEGF-R1 w ith NOS II (p = 0.0008) and NOS III (p = 0.011) The oedema volumes could no t be correlated significantly with NOS or VEGF-R1 expression values but wit h those of endothelial staining (p = 002). We observed a trend towards high er Ki-MIP expression values together with higher oedema volume extensions. In situ hybridization demonstrated reaction products in endothelial and per ivascular regions and sometimes scattered throughout the specimens revealin g 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 ce lls. The expression of NOS II in glioblastomas is restricted to infiltratin g macrophages. NOS II and III expressions were observed significantly toget her with that of VEGF-R1. Neither NOS I-III nor VEGF-R expression could be correlated with the extension of the peritumoral oedema.