title: Novel cell-free strategy for therapeutic angiogenesis: in vitro generated conditioned medium can replace progenitor cell transplantation.
abstract: BACKGROUND
  Current evidence suggests that endothelial progenitor cells (EPC) contribute to ischemic tissue repair by both secretion of paracrine factors and incorporation into developing vessels. We tested the hypothesis that cell-free administration of paracrine factors secreted by cultured EPC may achieve an angiogenic effect equivalent to cell therapy.
  
  
  RESULTS
  EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC subjected to 72 hours of hypoxia. In vitro, EPC-CM significantly inhibited apoptosis of mature endothelial cells and promoted angiogenesis in a rat aortic ring assay. The therapeutic potential of EPC-CM as compared to EPC transplantation was evaluated in a rat model of chronic hindlimb ischemia. Serial intramuscular injections of EPC-CM and EPC both significantly increased hindlimb blood flow assessed by laser Doppler (81.2+/-2.9% and 83.7+/-3.0% vs. 53.5+/-2.4% of normal, P<0.01) and improved muscle performance. A significantly increased capillary density (1.62+/-0.03 and 1.68+/-0.05/muscle fiber, P<0.05), enhanced vascular maturation (8.6+/-0.3 and 8.1+/-0.4/HPF, P<0.05) and muscle viability corroborated the findings of improved hindlimb perfusion and muscle function. Furthermore, EPC-CM transplantation stimulated the mobilization of bone marrow (BM)-derived EPC compared to control (678.7+/-44.1 vs. 340.0+/-29.1 CD34(+)/CD45(-) cells/1x10(5) mononuclear cells, P<0.05) and their recruitment to the ischemic muscles (5.9+/-0.7 vs. 2.6+/-0.4 CD34(+) cells/HPF, P<0.001) 3 days after the last injection.
  
  
  CONCLUSIONS
  Intramuscular injection of EPC-CM is as effective as cell transplantation for promoting tissue revascularization and functional recovery. Owing to the technical and practical limitations of cell therapy, cell free conditioned media may represent a potent alternative for therapeutic angiogenesis in ischemic cardiovascular diseases.
authors.affiliation: Department of Vascular Medicine, Swiss Cardiovascular Center, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland.
authors.forename: Stefano
authors.initials: S
authors.lastname: Di Santo
authors.forename: Zijiang
authors.initials: Z
authors.lastname: Yang
authors.forename: Moritz
authors.initials: M
authors.lastname: Wyler von Ballmoos
authors.forename: Jan
authors.initials: J
authors.lastname: Voelzmann
authors.forename: Nicolas
authors.initials: N
authors.lastname: Diehm
authors.forename: Iris
authors.initials: I
authors.lastname: Baumgartner
authors.forename: Christoph
authors.initials: C
authors.lastname: Kalka
mesh_terms: D000328:Adult; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D015687:Cell Hypoxia; D002460:Cell Line; D002470:Cell Survival; D002474:Cell-Free System; D017077:Culture Media, Conditioned; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D019650:Hematopoietic Stem Cell Mobilization; D006614:Hindlimb / Q000098:blood supply / Q000473:pathology / Q000503:physiopathology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D007511:Ischemia / Q000139:chemically induced / Q000473:pathology / Q000503:physiopathology; D009132:Muscles / Q000502:physiology; D018919:Neovascularization, Physiologic; D010477:Perfusion; D051381:Rats; D011923:Rats, Nude; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology*
chemical_list: D017077:Culture Media, Conditioned; D036341:Intercellular Signaling Peptides and Proteins