Proteomic Characterization of Vascular Endothelial Cell-Conditioned Medium Chemo-Attractive to Umbilical Cord Blood Monocytes: Implications for Angiogenesis in Response to Hypoxia.

Abstract 3061

Poster Board II-1037

At the onset of a wound, injured blood vessels and degranulating platelets release a number of growth factors and cytokines, and the blood clot forms of cross-linked fibrin and extracellular matrix proteins. Peripheral blood monocytes play a central role in angiogenesis. Monocyte migration toward injured tissue depending on a chemotactic gradient, as well as the activation and adherence of monocytes to the endothelial cell layer. Endothelial cells provide adhesion molecules and secreted proteins acting as monocyte chemoattractants and monocyte activators leading to transendothelial migration. Studies examined direct endothelial cell-monocyte interactions and cytokines produced by monocytes that enhance angiogenesis during wound healing, but the precise analyses of factors secreted by vascular endothelial cells in response to hypoxia have not been examined. This report identifies proteins that contribute to enhanced monocyte migration. To determine whether umbilical cord blood (UCB)-derived monocytes may serve to augment endothelial cell function, Matrigel experiments were conducted. HUVEC tubule formation in Matrigel in basal media was compared with HUVEC alone and HUVEC + UCB monocytes. We compared the kinetics and stability of enclosed endothelial cell networks formed by HUVEC. CD14+ monocytes were isolated via magnetic bead-labeling (AutoMACS) with purity >80% and cultured in RPMI 1640 medium with 5% FBS. UCB-derived monocytes significantly enhanced HUVEC tubule formation in Matrigel assays (e.g., with the addition of monocyte-conditioned media (CM) (137 networks) compared to non-CM (80 networks)). UCB monocyte-CM also improved the stability of the enclosed cell networks in culture as structures persisted beyond 24h, while none were present in the non-CM matrigel cultures. To determine whether UCB-derived monocytes respond to factors elicited by injured vascular endothelial cells, we measured UCB-derived monocyte chemotaxis to HUVEC-CM in hypoxic conditions (5% O₂) using Transwell plates (8.0 μm pores). Monocytes migrated for four hours to either media alone or HUVEC-CM. We observed a 3.3 fold increase in the migration of the monocytes to HUVEC-CM over that of basal media. To characterize the protein expression profiles and identify the molecules associated with monocyte migration response to endothelial cells maintained in a hypoxic environment, cellular proteins secreted by HUVEC in response to hypoxia were analyzed using mass spectrometry proteomics. Proteins were run on SDS-PAGE gels and after Coomassie staining, gel regions corresponding to MW < 25 KDa were excised and subjected to in-gel-trypsin-digestion. Peptides were analyzed by liquid chromatography mass spectroscopy (LC-MS). The mass spectrometer was operated in a data-dependant mode in which every high resolution FT full scan (resolution=60,000). Mass spectrometry raw files were searched against the Swiss-Prot human database and peptides with P values less than 0.01 were reported. The activity of every protein was determined through literature review, with an emphasis on roles pertaining to angiogenesis, inflammation, monocyte activation and chemotaxis, and transendothelial migration. Of 206 differentially expressed proteins, six were further studied and were Cyclophilin A, Cyr61 (CCN1), connective tissue growth factor (CCN2), pancreatic RNase A, macrophage migration inhibitory factor (MIG), and. Higher expression of all proteins (except thioredoxin) was confirmed by Western blot and ELISA. The exact mechanisms by which monocytes promote formation of new vasculature is not elucidated, but there is evidence of both direct contact in the activation of endothelial cells and by paracrine mechanisms secreting proangiogenic factors (VEGF, bFGF, PDGF, and TNFαa). We have identified other factors that may play a role. Our results support reported increased vascularization in a limb ischemia murine model treated with human CD14+ monocytes and support with molecular evidence that CD14+ monocytes increase the kinetics of collateral artery growth after acute femoral artery occlusion in animal models. In conclusion, these in vitro analyses confirm that UCB-derived monocytes significantly augment HUVEC function and the precise identification of proteins regulated in hypoxic HUVEC CM enhancing monocyte chemotaxis may have implications in diverse processes including wound healing.