Original article Soluble factors released by endothelial progenitor cells promote migration of endothelial cells and cardiac resident progenitor cells Carmen Urbich a , Alexandra Aicher a , Christopher Heeschen a , Elisabeth Dernbach a , Wolf K. Hofmann b , Andreas M. Zeiher a , Stefanie Dimmeler a, * a Molecular Cardiology, Department of Internal Medicine III, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany b Department of Hematology and Oncology, Internal Medicine II, University of Frankfurt, Theodor-Stern-Kai 7, Frankfurt, Germany Received 12 October 2004; received in revised form 7 June 2005; accepted 11 July 2005 Available online 29 September 2005 Abstract Circulating endothelial progenitor cells (EPC) are incorporated into newly formed capillaries, enhance neovascularization after hind limb ischemia and improve cardiac function after ischemic injury. Incorporated progenitor cells may also promote neovascularization and cardiac regeneration by releasing factors, which act in a paracrine manner to support local angiogenesis and mobilize tissue residing progenitor cells. Therefore, we analyzed the expression profile of cytokines in human peripheral blood-derived EPC as opposed to human umbilical vein endothelial cells (HUVEC), human microvascular endothelial cells (HMVEC), and CD14 + monocytes by microarray technology. A gene tree analysis revealed a distinct expression pattern of angiogenic growth factors in EPC, mature endothelial cells, and CD14 + monocytes. VEGF-A, VEGF-B, SDF-1, and IGF-1 mRNA levels were higher in EPC as compared to HUVEC or HMVEC. The enhanced mRNA expression was paralleled by a significant release ofVEGF, SDF-1, and IGF-1 protein into the cell culture supernatant of EPC. Moreover, immunohistological analysis of ischemic limbs from nude rats revealed that VEGF is also released from recruited human EPC in vivo. As a functional conse- quence, conditioned medium of EPC induced a strong migratory response of mature endothelial cells, which was significantly inhibited by VEGF and SDF-1 neutralizing antibodies. Finally, conditioned medium of EPC significantly stimulated the migration of cardiac resident c-kit + progenitor cells in vitro. Taken together, EPC exhibit a high expression of angiogenic growth factors, which enhanced migration of mature endothelial cells and tissue resident cardiac progenitor cells. In addition to the physical contribution of EPC to newly formed vessels, the enhanced expression of cytokines may be a supportive mechanism to improve blood vessel formation and cardiac regeneration after cell therapy. © 2005 Elsevier Ltd. All rights reserved. Keywords: Growth factors; Endothelial progenitor cells; Angiogenesis 1. Introduction Postnatal neovascularization is an important process to res- cue tissue from critical ischemia [1]. Circulating bone marrow-derived endothelial progenitor cells (EPC) signifi- cantly contribute to adult blood vessel formation [2–4]. These endothelial progenitor cells are positive for endothelial marker proteins such as VEGF receptor 2 (KDR), von Willebrand factor (vWF), VE-cadherin, eNOS, take-up of diacetylated LDL, and bind lectin [5–7]. Injected EPC were shown to inte- grate into blood vessels and improve neovascularization of ischemic hind limbs and ischemic hearts in animal models [8–11]. In accordance, initial clinical trials indicate that bone marrow-derived or circulating blood-derived progenitor cells may be therapeutically useful to improve blood flow of ischemic tissue and improve heart function [12,13]. At present it is unclear, whether the improved heart function seen in patients after progenitor cell therapy is exclusively mediated by an improvement of neovascularization or also may reflect regeneration of cardiac tissue. Indeed, EPC have been shown to acquire a cardiac phenotype in vitro after co-cultivation * Corresponding author. Tel.: +49 69 6301 7440/5789; fax: +49 69 6301 7113/6374. E-mail address: Dimmeler@em.uni-frankfurt.de (S. Dimmeler). Journal of Molecular and Cellular Cardiology 39 (2005) 733–742 www.elsevier.com/locate/yjmcc 0022-2828/$ - see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2005.07.003