Bone marrow (BM)-derived circulating endothelial precursor cells (CEPs) are thought to play a role in postnatal angiogenesis. Emerging evidence suggests that angiogenic stress of vascular trauma may induce mobilization of CEPs to the peripheral circulation. In this regard, we studied the kinetics of CEP mobilization in two groups of patients who experienced acute vascular insult secondary to burns or coronary artery bypass grafting (CABG). In both burn and CABG patients, there was a consistent, rapid increase in the number of CEPs, determined by their surface expression pattern of vascular endothelial growth factor receptor 2 (VEGFR2), vascular endothelial cadherin (VE-cadherin), and AC133. Within the first 6 to 12 hours after injury, the percentage of CEPs in the peripheral blood of burn or CABG patients increased almost 50-fold, returning to basal levels within 48 to 72 hours. Mobilized cells also formed late-outgrowth endothelial colonies (CFU-ECs) in culture, indicating that a small, but significant, number of circulating endothelial cells were BM-derived CEPs. In parallel to the mobilization of CEPs, there was also a rapid elevation of VEGF plasma levels. Maximum VEGF levels were detected within 6 to 12 hours of vascular trauma and decreased to baseline levels after 48 to 72 hours. Acute elevation of VEGF in the mice plasma resulted in a similar kinetics of mobilization of VEGFR2(+) cells. On the basis of these results, we propose that vascular trauma may induce release of chemokines, such as VEGF, that promotes rapid mobilization of CEPs to the peripheral circulation. Strategies to improve the mobilization and incorporation of CEPs may contribute to the acceleration of vascularization of the injured vascular tissue.

In response to endotoxin, macrophages secrete a protein with a molecular mass of %6000 Da and with an affinity for heparin. This protein, which we term "macrophage inflammatory protein 2," is a potent chemotactic agent for human polymorphonuclear leukocytes. In addition, subcutaneous administration of the monokine causes a localized inflammatory reaction. Partial N-terminal sequence data reveal similarity to a family of proteins, the archetype of which is platelet factor 4. Although macrophage inflammatory protein 2 is a distinct member ofthe platelet factor 4 family, its sequence is most closely related to that of the gro/KC gene product, which is expressed in transformed or platelet-derived growth factortreated cells.One hallmark of the acute inflammatory state is the recruitment and activation of polymorphonuclear leukocytes (PMNs). Numerous mediators have been shown to be involved in this process, including leukotrienes (1, 2); complement components (3, 4); cachectin/tumor necrosis factor (TNF) (5-7); bacterial products (4,8); neutrophil-activating protein 1 (NAP-1), a recently described monokine with sequence relatedness to the platelet factor 4 (PF4) family (9)(10)(11)(12)(13)(14)(15); and another monokine, macrophage inflammatory protein 1 (MIP-1) (16).We have previously shown that endotoxin-stimulated macrophages secrete two proteins that bind to heparin-Sepharose and elute only with high-salt buffer (16 Research, Leuven, Belgium), and purified human NAP-1 protein was given by T. Yoshimura and E. Leonard (National Cancer Institute, Bethesda, MD). All other reagents were obtained from Sigma.Cell Culture. The mouse macrophage cell line RAW 264.7 was obtained from American Type Culture Collection. The cells were maintained in culture and stimulated with endotoxin to produce conditioned medium as previously described (16).Purification of MIP-2. MIP-2 was purified by using methodology previously described for MIP-1 (16). The degree of purification was followed by SDS/PAGE with silver staining. In brief, 2 liters of conditioned supernatant from endotoxinstimulated RAW 264.7 cells were concentrated and diafiltrated against 20 mM Tris HCl (pH 8.0) and applied to a Mono Q 10/10 (anion-exchange) column (Pharmacia LKB Biotechnology, Rahway, NJ). Greater than 90% of the MIP-2 was observed not to bind to the column and was recovered in the effluent.The peak MIP-2-containing fractions were applied to a heparin-conjugated Sepharose (Pharmacia LKB Biotechnology) column equilibrated with 20 mM Tris HCl (pH 8.0) and eluted with a 0-2 M NaCl linear gradient in the same buffer. MIP-2 eluted at -0.75 M NaCl. Peak fractions were concentrated in a Centricon ultrafiltration device with a molecular weight cutoff of 3000 (Amicon, Danvers, MA) and applied to a Superose 12 (gel-filtration; Pharmacia LKB Biotechnology) column equilibrated with 100 mM ammonium acetate. From 2 liters of RAW 264.7 conditioned medium (which equaled -100 mg total protein), we generally isolated 0.5 mg of MIP-2 as assessed by the Bradford protein...

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