Angiogenic effects of stromal cell-derived factor-1 (SDF-1/CXCL12) variants in vitro and the in vivo expressions of CXCL12 variants and CXCR4 in human critical leg ischemia

J Vasc Surg. 2010 Mar;51(3):689-99. doi: 10.1016/j.jvs.2009.10.044.


Purpose: Critical leg ischemia (CLI) is associated with a high morbidity and mortality. Therapeutic angiogenesis is still being investigated as a possible alternative treatment option for CLI. CXCL12, a chemokine, is known to have two spliced variants, CXCL12alpha and CXCL12beta, but the significance remains unknown. The study investigated the angiogenic effects of CXCL12, protein expressions of CXCL12, and the receptor CXCR4 in human CLI.

Methods: In vitro, human microvascular endothelial cells (HMEC-1) were used. Cell proliferation was assessed using methylene blue assay and cell count method. Apoptosis was determined by counting the pyknotic nuclei after 4'-6-diamidino-2-phenylindole staining and confirmed by caspase-3 assay. We employed matrigel as capillary tube formation assay. The activity of signaling pathways was measured using Western blotting. In vivo, gastrocnemius biopsies were obtained from the lower limbs of patients with CLI and controls (n = 12 each). Immunohistochemistry, double immunofluorescence labeling, and Western blotting were then performed.

Results: CXCL12 attenuated HMEC-1 apoptosis (P < .01), stimulated cell proliferation (P < .05) and capillary tube formation (P < .01). Compared with CXCL12alpha, CXCL12beta has a greater effect on apoptosis and cell proliferation (P < .01). Treatment with both variants resulted in time-dependent activation of PI3K/Akt and p44/42 but not p38 MAP kinase. In CLI, CXCL12alpha was expressed by skeletal muscle fibers with minimal expression of CXCL12beta. CXCR4 was extensively expressed and colocalized to microvessels. A significant 2.6-fold increase in CXCL12alpha and CXCR4 expressions (P < .01) were noted in CLI but not for CXCL12beta (P > .05).

Conclusions: The study showed that CXCL12beta had more potent angiogenic properties but was not elevated in human CLI biopsies. This provided an interesting finding on the role of CXCL12 variants in pathophysiologic angiogenic response in CLI.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aged
  • Angiogenesis Inducing Agents / pharmacology
  • Apoptosis
  • Biopsy
  • Blotting, Western
  • Case-Control Studies
  • Cell Proliferation
  • Cells, Cultured
  • Chemokine CXCL12 / metabolism*
  • Chemokine CXCL12 / pharmacology
  • Critical Illness
  • Dose-Response Relationship, Drug
  • Endothelial Cells / drug effects
  • Endothelial Cells / immunology*
  • Endothelial Cells / pathology
  • Female
  • Fluorescent Antibody Technique
  • Humans
  • Ischemia / immunology*
  • Ischemia / pathology
  • Ischemia / physiopathology
  • Lower Extremity
  • Male
  • Middle Aged
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Mitogen-Activated Protein Kinase 3 / metabolism
  • Muscle, Skeletal / blood supply*
  • Muscle, Skeletal / immunology
  • Neovascularization, Physiologic* / drug effects
  • Phosphatidylinositol 3-Kinases / metabolism
  • Protein Isoforms
  • Proto-Oncogene Proteins c-akt / metabolism
  • Receptors, CXCR4 / metabolism*
  • Signal Transduction
  • Time Factors


  • Angiogenesis Inducing Agents
  • CXCL12 protein, human
  • CXCR4 protein, human
  • Chemokine CXCL12
  • Protein Isoforms
  • Receptors, CXCR4
  • Phosphatidylinositol 3-Kinases
  • Proto-Oncogene Proteins c-akt
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3