Sprouting angiogenesis is associated with extensive extracellular matrix (ECM) remodeling. The molecular mechanisms involved in building the vascular microenvironment and its impact on capillary formation remain elusive. We therefore performed a proteomic analysis of ECM from endothelial cells maintained in hypoxia, a major stimulator of angiogenesis. Here, we report the characterization of lysyl oxidase-like protein-2 (LOXL2) as a hypoxia-target expressed in neovessels and accumulated in the endothelial ECM. IntroductionAngiogenesis occurs during development and tissue remodeling, and in the pathologic context of cardiovascular ischemic diseases or tumor growth. Sprouting of new vessels is initiated by stimulation of endothelial cells (ECs) by a combination of signals from the microenvironment that includes oxygen tension and growth factors. Cells from not yet vascularized tissue and ECs that invade this microenvironment are both hypoxia targets through the Hypoxia Inducible Factor (HIF) pathway. HIF activates transcription of genes coding for autocrine/paracrine factors like vascular endothelial growth factor (VEGF) and extracellular matrix (ECM) components. 1 Synergy between these responses is assumed through local concentration of growth factors in the ECM where they function as attractant for ECs. Specialized ECs, called tip cells, lead vascular growth by sending out filopodia to explore the hypoxic microenvironment. 2 Tip cells are thus continuously exposed to low oxygen concentration. 3 Stalk cells, located behind the tip cells, serve to vessel growth by proliferation, lumen formation and junction establishment. 4 Vascular ECM undergoes major remodeling during angiogenesis, consisting in ECM/basement membrane degradation, provisional ECM generation and assembly of a new basement membrane. ECM-mediated mechanotransductive signaling regulates 3D multicellular organization, including lumen formation and tubulogenesis. Thus, in addition to storing angiogenic factors and providing structural features, ECM is a dynamic promoter of angiogenesis. 5 Subendothelial basement membrane is composed of nonfibrillar collagen IV, laminin, perlecan and nidogens. Other collagens (VIII, XV, and XVIII), fibronectin and matricellular proteins (thrombospondin-1, Cyr61) are associated with the basement membrane. There is only little data concerning the assembly of the vascular microenvironment and its impact on angiogenesis. Genes encoding ECM components and enzymes involved in their assembly and stabilization are targets of hypoxia in ECs. 1 To identify key regulators of the angiogenesis-associated ECM remodeling, we performed a proteomic analysis of endothelial ECM generated in vitro under hypoxic conditions. We found that the ECM cross-linking enzyme, lysyl oxidase-like protein-2 (LOXL2) is a major target of hypoxia. Lysyl oxidases, consisting in lysyl oxidase (LOX) and 4 lysyl oxidase-like proteins (LOXL 1 to 4), catalyze the deamination of lysines and hydroxylysines, generating aldehydes that spontaneously react to form co...
The increased incidence of autoantibodies in malignancies has been described since the 1970s. Thus the ability to determine molecular fingerprinting of autoantibodies (antibody signatures) may provide useful clinical diagnostic and prognostic information. This review describes the use of several proteomics approaches for the identification of antigens recognized by these autoantibodies. Serological proteome analysis combines separation of tumor cell proteins on two-dimensional gel electrophoresis gels, Western blotting with sera of patients and healthy subjects, and identification of the detected antigens by MS. Alternatively multiple affinity protein profiling combines isolation of the antigens recognized by patient antibodies by two-dimensional immunoaffinity chromatography and identification by MS/MS. The use and limitations of reverse phase protein microarrays for testing patient serum containing autoantibodies are also considered. Lastly
Galectin 1 (GAL1) is a beta-galactoside-binding lectin involved in cell cycle progression. GAL1 overexpression is associated with neoplastic transformation and loss of differentiation. The gene encoding for human GAL1 resides on chromosome 22(q12; q13), and its expression is developmentally regulated. Although devoid of signal peptide GAL1 can be externalized from cells by a mechanism independent of the normal secretory process. We report here on a study of the effects of erythroid differentiation of the human leukemia cell line K562 on GAL1 protein expression. In undifferentiated K562 cells, GAL1 was expressed into the cytosol. However, the amount of GAL1 was surprisingly weaker in K562 cells than in other leukemia cell lines such as TF-1 or KG1a. Treatment of K562 cells with erythropoietin (EPO) or with aphidicolin (APH), an inhibitor for DNA polymerase alpha, induced an erythroid phenotype and led to the externalization of cytosolic GAL1 which was then bound to ligands on cell surface in a galactoside-inhibitable fashion. Our results demonstrate that acquisition of an erythroid phenotype is associated with an externalization of GAL1. The autocrine binding of GAL1 to cell surface ligands of non adherent cells such as K562 suggest that GAL1 is implicated rather in signal transduction than in cell-cell or cell-matrix interaction. Moreover, the reciprocal translocation involving chromosomes 9 and 22 t(9;22) present in K562 cells might explain the weak expression of GAL1 in K562 leukemia cells.
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