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, CXCL12α and CXCL12β, 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 CXCL12α, CXCL12β 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, CXCL12α was expressed by skeletal muscle fibers with minimal expression of CXCL12β. CXCR4 was extensively expressed and colocalized to microvessels. A significant 2.6-fold increase in CXCL12α and CXCR4 expressions (P < .01) were noted in CLI but not for CXCL12β (P > .05).

Conclusions

The study showed that CXCL12β 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.

Clinical Relevance

The in vitro study showed that CXCL12β had more potent angiogenic properties compared to CXCL12α, and both of these act via the p44/42 and PI3K/Akt pathways. The in vivo data using tissues of human CLI confirmed the pathophysiological changes that showed deficient CXCL12β and the increased expression of CXCR4 by microvessels, suggesting that CXCL12 plays an important role in human CLI. Therefore, the use of CXCL12β as a proangiogenic agent may be more likely to provide encouraging results in future experiments and possibly in the use as a possible therapeutic angiogenic agent.