A Role for Activation of the Kallikrein-Kinin System in the Homing Property of Endothelial Progenitor Cells.

The plasma kallikrein–kinin system (KKS) plays an important role in inflammation, thrombosis and vascular pathology. Activation of the KKS results in bradykinin release, which increases vascular permeability and modulates inflammatory responses. Circulating endothelial progenitor cells (EPCs) home to ischemic and inflamed tissues and participate in the vasculogenesis. However, the homing mechanism remains poorly understood. We now report that activation of the KKS regulates the homing property of EPCs using in vivo and in vitro approaches. We have previously shown that the Lewis rat, which displays a mutant kininogen (Ser511Ala), is susceptible to peptidoglycan-polysaccharide (PG-PS)- induced systemic inflammation including arthritis and displays activation of the KKS and increased bradykinin levels. CD34-positive EPCs were isolated from bone marrow of disease-free Lewis rats by Ficoll gradient centrifugation. After culture in EGM2 on collagen surfaces for 7 days, a single colony with cobblestone-like morphology appeared and was subjected to large-scale expansion. Compared with differentiated rat lung microvessel endothelial cells (rLMECs), rat EPCs expressed similar levels of endothelial cell lineage marker mRNA such as VE-cadherin, CD31, vWF and uPAR. EPCs exclusively expressed hematopoietic progenitor cell markers, Sca-1 and CD34. Both of EPCs and rLMECs expressed bradykinin type 2 receptor (B2R) but not B1R. To observe the in vivo mobilization of EPCs, EPCs were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) and injected into Lewis rats in which inflammation/arthritis was induced by PG-PS. Two weeks after PG-PS injection, pannus formed in the synovial tissues, representing the entry into the chronic phase of arthritis. The fluorescently-labeled EPCs were selectively detected in the inflamed synovium of PG-PS-treated rats, but not that of disease-free rats. As detected by confocal microscopy, implanted EPCs formed new blood vessels in the inflamed synovial tissues. To further investigate the mechanism by which the KKS regulates EPC mobilization, we observed that bradykinin (a B2R agonist), but not des-Arg9-bradykinin (a B1R agonist), stimulated EPC transmigration through confluent rLMEC layer at 100 and 1000 nM (P<0.01 and P<0.001, respectively). Bradykinin did not affect EPC adhesion to rLMECs. Priming by tumor necrosis factor a (TNFa), a major inflammatory cytokine, induced transendothelial migration of EPCs by 100 nM des-Arg9- bradykinin (168.56 ± 9.56 vs 96.02 ± 10.23, P<0.001). The priming effect resulted from TNFa induction of B1R mRNA. As determined by RT-PCR, rat EPCs expressed mRNA of multiple homing receptors including chemokine (C-X-C motif) receptor 4, CD49d and CD62-E, but not CD11a. Thus, activation of the KKS regulates the homing property of EPCs, which constitutes an additional mechanism for involvement of the KKS in the vasculogenesis. B2R predominantly mediates transendothelial migration of normal EPCs, whereas B1R seems responsible for that of inflamed EPCs. Kinins and cytokines such as TNFa may synergize to regulate the mobilization of EPCs. These observations provide a new insight into understanding the role of the KKS in vascular pathology and inflammation.