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

Abstract 1487

Peripheral blood monocytes play a central role in angiogenesis in a variety of physiologic and pathologic processes. To determine whether UCB-derived monocytes respond to factors elicited by injured vascular endothelial cells, we measured UCB-derived monocyte chemotaxis to human umbilical vein endothelial cells (HUVEC)-conditioned media in hypoxic conditions (5% O₂). HUVECs in endothelial growth media (EGM) containing HGF, IGF, FGF, heparin, EGF) media were grown until confluent under normoxic conditions. At confluence, HUVEC grown in the EGM media were washed and replaced with 1640 RPMI/5% FBS media. HUVEC were exposed to hypoxic conditions (1% O₂) for 24 hours. The CM was collected, centrifuged at 12,000x g removing cellular debris, aliquoted, and stored at -20°C. Migration experiments were conducted using Transwell plates. UCB was obtained 24–48 hrs following delivery and mononuclear cells (MNC) were isolated by centrifugation through a Ficoll-Paque density gradient. UCB CD14⁺ cells were selected by magnetic labeling and sorting using AutoMACS magnetic cell sorter (Miltenyi, CA). UCB monocytes (3.0 × 10⁵ cells/well) were placed in 8.0 μm upper transwells and the lower well contained CM, antibody-depleted CM, or fresh 1640 RPMI/5% FBS basal media. Monocytes were incubated above the various CM for 2 hours; afterwards cells in the lower well were labeled with anti-CD14⁺ APC antibodies and analyzed using flow cytometry (BD Canto II or BD LSRII, BD Biosciences). The relative migration index was measured examined the number of CD45⁺ CD14⁺ cells collected in 90 seconds compared to aliquots of 3.0 ×10⁵ cells corresponding to 100%. We observed a 3.3 fold increase in the migration of the monocytes to HUVEC-conditioned media over that of basal media. To characterize the protein expression profiles and identify the molecules associated with monocyte migration response to hypoxic HUVEC, the cellular proteins secreted by HUVEC in response to hypoxia were analyzed using mass spectrometry proteomics. Of 206 differentially expressed proteins, six were further studied due to known function in angiogenesis and inflammation including: cyclophilin A, Cyr61 (CCN1), connective tissue growth factor (CCN2), pancreatic RNase A, macrophage migration inhibitory factor (MIF), and thioredoxin. Next, to examine the functional contribution of each identified secreted factors, HUVEC-CM aliquots were incubated with anti-cyclophilin A, anti-CCN1, anti-CCN2, anti-MIF, anti-thioredoxin, and anti-VEGF antibodies then mixed with protein A/G beads followed by centrifugation of beads and removal of supernatant. Random migration for monocytes in the transwell assay was found to be 6.3± 4.7 % compared to conditioned media. Relative monocyte migration to various antibody depleted-CM compared to non-depleted CM showed that monocytes had a reduced migration of 14.7 ± 2.7% with cyclophilin A-depleted media; 6.2 ± 0.4% with CCN2-depleted media; 11.5 ± 0.95% with CCN1-depleted media; 11.3 ± 0.5% to MIF-depleted media; 9.2 ± 3.2% thioredoxin-depleted media; 13.4 ± 0.2% VEGF-depleted media; and 9.18% to conditioned media depleted containing antibodies directed against all of the above factors. Besides VEGF, several other factors released by endothelial cells under hypoxic conditions expected in a wound area, function as chemoattractants for monocytes. While anti-VEGF neutralized media reduced migration by about 13%, cyclophilin A-depleted media reduced migration by about 15% and other protein depletions reduced migration by as much as 11%. These results suggest that HUVEC produce additional monocyte chemoattractants besides VEGF in particular cyclophilin A which affects migration more than VEGF. Although previous studies have examined direct endothelial cell – monocyte interactions and cytokines produced by monocytes that enhance angiogenesis during wound healing, precise analyses of factors secreted by injured vascular endothelial cells in response to hypoxia have not to our knowledge been examined. This report is the first to identify these proteins that contribute to enhanced monocyte migration.