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

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, CXCL12 alpha and CXCL12 beta, but the significance remains unknown. The study investigated the angiogenic effects of CXCL12, protein expressions of CXCL12, and the receptor CXCR4 in human CIA. 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 immunoflorescence 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 CXCL12 alpha, CXCL12 beta has a greater effect oil 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 CXCL12 alpha was expressed by skeletal muscle fibers with minimal expression of CXCL12 beta. CXCR4 was extensively expressed and colocalized to microvessels. A significant 2.6-fold increase in CXCL12 alpha and CXCR4 expressions (P < .01) were noted in CLI but not for CXCL12 beta (P > .05). Conclusions:The study showed that CXCL12 beta had more potent angiogenic properties but was not elevated ill human CLI biopsies. This provided all interesting finding oil the role of CXCL12 variants in pathophysiologic angiogenic response in CLI. (J Vasc Surg 2010;51:689-99.)