IPVH10100 MilliporeImmobilon^®-P PVDF Membrane

ABSTRACT: INTRODUCTION: Immunological studies of giant cell arteritis (GCA) suggest that a triggering antigen of unknown nature could generate a specific immune response. We thus decided to detect autoantibodies directed against endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in the serum of GCA patients and to identify their target antigens. METHODS: Sera from 15 GCA patients were tested in 5 pools of 3 patients' sera and compared to a sera pool from 12 healthy controls (HCs). Serum immunoglobulin G (IgG) reactivity was analysed by 2-D electrophoresis and immunoblotting with antigens from human umbilical vein ECs (HUVECs) and mammary artery VSMCs. Target antigens were identified by mass spectrometry. RESULTS: Serum IgG from GCA patients recognised 162 ± 3 (mean ± SD) and 100 ± 17 (mean ± SD) protein spots from HUVECs and VSMCs, respectively, and that from HCs recognised 79 and 94 protein spots, respectively. In total, 30 spots from HUVECs and 19 from VSMCs were recognised by at least two-thirds and three-fifths, respectively, of the pools of sera from GCA patients and not by sera from HCs. Among identified proteins, we found vinculin, lamin A/C, voltage-dependent anion-selective channel protein 2, annexin V and other proteins involved in cell energy metabolism and key cellular pathways. Ingenuity pathway analysis revealed that most identified target antigens interacted with growth factor receptor-bound protein 2. CONCLUSIONS: IgG antibodies to proteins in the proteome of ECs and VSMCs are present in the sera of GCA patients and recognise cellular targets that play key roles in cell biology and maintenance of homeostasis. Their potential pathogenic role remains to be determined.

Most proangiogenic polypeptide growth factors and chemokines enhance vascular permeability, including vascular endothelial growth factor (VEGF), the main target for anti-angiogenic-based therapies, and interleukin-8 (IL-8), a potent proinflammatory mediator. Here, we show that in endothelial cells IL-8 initiates a signaling route that converges with that deployed by VEGF at the level of the small GTPase Rac1 and that both act through the p21-activated kinase to promote the phosphorylation and internalization of VE-cadherin. However, whereas VEGF activates Rac1 through Src-related kinases, IL-8 specifically signals to Rac1 through its cognate G protein-linked receptor, CXCR2, and the stimulation of the phosphatidylinositol 3-kinase gamma (PI3Kgamma) catalytic isoform, thereby providing a specific molecular targeted intervention in vascular permeability. These results prompted us to investigate the potential role of IL-8 signaling in a mouse model for retinal vascular hyperpermeability. Importantly, we observed that IL-8 is upregulated upon laser-induced retinal damage, which recapitulates enhanced vascularization, leakage, and inflammatory responses. Moreover, blockade of CXCR2 and PI3Kgamma was able to limit neovascularization and choroidal edema, as well as macrophage infiltration, therefore contributing to reduce retinal damage. These findings indicate that the CXCR2 and PI3Kgamma signaling pathway may represent a suitable target for the development of novel therapeutic strategies for human diseases characterized by vascular leakage.