Urinary proteomics and the role of orosomucoid (ORM) in vascularization of bladder cancer
2-dimensional gel electrophoresis (2-DE) is currently the method of choice for separation of complex protein mixtures such as in cell and tissue extracts or body fluids including urine or serum. Human urine plays a central role in clinical diagnostics of diseases such as cancer and inflammation. Researchers and scientists are working on the development of a map of the human proteome but in the literature there are only a few investigations about urinary proteomics and their behaviour in the case of bladder tumor. The first aim of this study was to view the whole proteins present in urine and to predict their functions. For this aim, fourtyfive urine samples from patients with bladder cancer of different stages, patients on follow-up and healthy volunteers were analysed by 2-DE. At the beginning of this study the 2-DE protein pattern of human urine was almost unknown because of lack of appropriate preparation of urine samples prior to the 2-DE. Thus, the study focused on methods for preparation of urine samples providing the best preservation of urinary proteins. After experimental optimization of 2-DE, usable 2-D protein patterns of urine were analysed to identify proteins related to bladder cancer using subsequent mass spectrometric analyses and/or conventional immunoblotting and immunohistochemical methods. In comparison to other cancer types, the bladder carcinoma is the seventh causing death in man and ninth in women among malignant tumors. The growth and metastasis as well as the transformation of superficial noninvasive tumors to an invasive tumor phenotype are closely associated with activation of angiogenesis resulting in neovascularisation of tumor tissue. This process is regulated by a net balance between angiogenic activators and inhibitors. In the present study, orosomucoid (ORM) and human zinc-alpha-2-glycoprotein (ZAG) have been identified to be increased in 2-DE of urine samples of patients with bladder cancer in comparison to the urine samples of healthy volunteers. Immunohistochemical results let assume that in addition to cancer cells also a part of the tissue resident inflammatory cells and endothelial cells of tumor associated blood vessels may serve as source for this increase of ORM in urine samples of patient with bladder cancer. ORM is an acute phase protein and increased in acute infection, inflammation, and cancer. Recent studies show that ORM forms a complex with the active form of plasminogen activator inhibitor-1 (PAI-1) in thymosin β4 (Tβ4)- activated but not in quiescent endothelial cells. Therefore it was aimed to determine a potential role of ORM up-regulation in angiogenesis. The findings presented in this study and results reported by Sorensson showed, that, in addition to the cancer cells, human vascular endothelial cells (HDMECs) produce ORM endogenously. For functional characterization of ORM in vascular endothelial cells, ORM-gene overexpression and ORM-gene silencing were perfromed. Employing the supernatants of HDMECs-ORM and HDMECs-ORM-siRNA in in-vitro angiogenesis assays, it was found that ORM supports the VEGF-induced endothelial tube formation. This supportive effect of ORM was potentiated by co-treatment of HDMECs with VEGF, ORM and anti-PAI-1 antibody. This study demonstrates for the first time that ORM is increased in urine of patients with bladder cancer and acts pro-angiogenic supporting the tube forming effects of VEGF. This supportative effect was significantly increased by additive blockage of PAI-1. In summary, the interaction between ORM and PAI-1 and anti-PAI-1 system seems to be essentially involved in the VEGF-mediated angiogenesis and probably in the vascularisation of urinary bladder cancer.