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Paracrine function of circulating angiogenic cells (CACs) is thought to contribute to vascular maintenance. We previously identified S100A8 and S100A9 secreted from physically inactive individuals' CD34 - /CD31 + CACs as negative regulators of capillary-like network formation. The purpose of this study was to investigate further the extremes of the continuum of CAC paracrine actions using two distinctly different groups representing "healthy" and "impaired" CAC function. We aimed to determine how capillary-like network formation in human umbilical vein endothelial cells (HUVECs) is affected by S100A8 and S100A9 in concentrations secreted by CACs from different ends of the health spectrum. CD34 - /CD31 + CACs were isolated and cultured from 10 impaired function individuals defined as older (50-89 yr), non-ST-elevation myocardial infarction patients and 10 healthy individuals defined as younger (18-35 yr), healthy individuals, and conditioned media (CM) was generated. CM from the impaired function group's CACs significantly diminished network formation compared with CM from the healthy group (P < 0.05). We identified elevations in S100A8, S100A9, and S100A8/A9 in the CM from the impaired function group (P < 0.05). Pretreatment of HUVECs with inhibitors to a known S100A8 and S100A9 receptor, Toll-like receptor 4 (TLR4), but not receptor for advanced glycation end products, improved HUVEC network formation (P < 0.05) compared with CM alone in the impaired function conditions. Exposure of HUVECs to the TLR4 signaling inhibitor also blocked recombinant S100A8- and S100A9-mediated reductions in network formation. Collectively, the results suggest that the mechanisms behind impaired CAC CD34 - /CD31 + CM-mediated reductions in capillary-like network formation involve secretion of S100A8 and S100A9 and binding of these proteins to TLR4 receptors on HUVECs., New & Noteworthy: S100A8 and S100A9 proteins in concentrations secreted by CD34 - /CD31 + circulating angiogenic cells (CACs) with impaired function reduce endothelial cell capillary-like network formation. These effects appear to be mediated by Toll-like receptor 4 and are absent with S100A8 and S100A9 in concentrations secreted by healthy CD34 - /CD31 + CACs., (Copyright © 2017 the American Physiological Society.)

We aimed to determine if chronic endurance-exercise habits affected redox status and paracrine function of CD34(+) and CD34(-)/CD31(+) circulating angiogenic cells (CACs). Subjects were healthy, nonsmoking men and women aged 18-35 yr and categorized by chronic physical activity habits. Blood was drawn from each subject for isolation and culture of CD34(+) and CD34(-)/CD31(+) CACs. No differences in redox status were found in any group across either cell type. Conditioned media (CM) was generated from the cultured CACs and used in an in vitro human umbilical vein endothelial cell-based tube assay. CM from CD34(+) cells from inactive individuals resulted in tube structures that were 29% shorter in length (P < 0.05) and 45% less complex (P < 0.05) than the endurance-trained group. CD34(-)/CD31(+) CM from inactive subjects resulted in tube structures that were 26% shorter in length (P < 0.05) and 42% less complex (P < 0.05) than endurance-trained individuals. Proteomics analyses identified S100A8 and S100A9 in the CM. S100A9 levels were 103% higher (P < 0.05) and S100A8 was 97% higher in the CD34(-)/CD31(+) CM of inactive subjects compared with their endurance-trained counterparts with no significant differences in either protein in the CM of CD34(+) CACs as a function of training status. Recombinant S100A8/A9 treatment at concentrations detected in inactive subjects' CD34(-)/CD31(+) CAC CM also reduced tube formation (P < 0.05). These findings are the first, to our knowledge, to demonstrate a differential paracrine role in CD34(+) and CD34(-)/CD31(+) CACs on tube formation as a function of chronic physical activity habits and identifies a differential secretion of S100A9 by CD34(-)/CD31(+) CACs due to habitual exercise., (Copyright © 2015 the American Physiological Society.)