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

Effects on cell proliferation by CXCL12α and CXCL12β. A, Dose-response curve showing the optimum dose of 100 ng/mL was used for CXCL12α and CXCL12β. B, Cell count method demonstrated increased cell number to 133 ± 12.33 × 10³ by CXCL12α and 156.17 ± 19.13 × 10³ by CXCL12β on day 3 of proliferation (P < .05). This is further confirmed by methylene blue assay (C), with the optical density of 0.87 ± 0.07 and 1.05 ± 0.05 for cells treated with CXCL12α and CXCL12β, respectively. In both methods, the increase in cell number to CXCL12β was significantly higher compared with that of CXCL12α (P < .01). D, The addition of U0126 (U) and PD98059 (PD) abrogated cell proliferation (P < .01), indicating that cell proliferation was stimulated by CXCL12α and CXCL12β via the p44/42 signaling pathway. Values expressed as mean ± standard deviation (SD). Error bars represent SD.

Antiapoptosis effects of CXCL12α and CXCL12β on HMEC-1. A, HMEC-1 underwent apoptosis after 24 hours of serum deprivation. The apoptotic cells were demonstrated by dense white nucleus after staining with DAPI. B, The percentage of apoptotic cells treated by CXCL12α was 12.49 ± 2.69%, and CXCL12β-treated cells had apoptosis of 9.77 ± 1.58%. Both of these were significantly lower compared with untreated cells in EBM 28.2 ± 4.3 (P < 0.01). In addition, the effect of CXCL12β was observed to be more significant compared with CXCL12α (P < .01). C, Western blot analysis of caspase-3 activation revealed that both CXCL12α and CXCL12β have antiapoptotic effect, with CXCL12β having a greater effect (P < .01). Bar graphs in relative density unit, RDU; EBM 0.21 ± 0.04 vs CXCL12α 0.66 ± 0.07 vs CXCL12β 0.27 ± 0.04. Full-length and activated fragments of the caspase-3 were seen at molecular weights of 35 kDa and 17 kDa, respectively. Jurkat cells treated with 25 μM etoposide were used as a molecular weight control. GAPDH was used as a loading control. D, Antiapoptotic effects were reversed with the addition of wortmannin (W) and LY294002 (LY), both PI3K inhibitors (P < .01). Therefore, the antiapoptotic effects of both CXCL12α and CXCL12β were via the PI3K/Akt pathway. Values are expressed as mean ± SD. Error bars represent standard deviation. Scale bar = 100 μm.

Effects of CXCL12α and CXCL12β on capillary tube formation. A, HMEC-1 formed capillary tubes on matrigel assay after 24-hour culture in 10% FCS, CXCL12α, and CXCL12β. B, Both CXCL12α and CXCL12β promoted capillary tube formations (P < .05). Although the area of tube network was higher in cells treated with CXCL12β, this was not found to be statistically significant compared with CXCL12α (Mean OD CXCL12α, 6.22 ± 1.58 vs CXCL12β 7.19 ± 1.71, P = .29). C, CXCL12-induced angiogenesis was inhibited by the addition of U0126 and PD98059 as well as wortmannin and LY294002 (P < .01). Values are expressed as mean ± SD. Error bars represent standard deviation. Scale bar = 50 μm.

Time-dependent activation of signaling pathways. A, Western blot analysis showed that the activation of p44/42 and Akt reached maximum around 10 minutes after treatment with CXCL12α and CXCL12β and returned to basal activity by 24 hours. The p38 signaling pathway was not activated. Total protein for p38, Akt, and p44/42 confirmed equal sample loading in each well. The bar graphs showed the relative amount of phospho-p38, Akt, and p44/42 to the corresponding total proteins measured in %RDU. Grey bar represented stimulation by CXCL12α, and white bar represented stimulation by CXCL12β. B, To verify specificity of inhibitors used, cells were treated with CXCL12α/β for 10 minutes in the presence or absence of inhibitors. Protein extracts were then prepared and subjected to Western blot analysis with antibodies to phospho–p38/Akt/p44/42 and total–p38/Akt/p44/42. NS, nonstimulated cells at 0 min; RDU, relative density unit; PD, PD98059; U, U0126; W, Wortmannin; LY, LY294002; SB, SB203580.

In vivo CXCL12 and CXCR4 expressions in CLI. CXCL12α was widely expressed in skeletal muscle fibers, A (i), but CXCL12β expression was not detected on the sections, A (ii). The distribution of CXCR4 expressions corresponded with that of microvessels, A (iii). Immunofluorescence double-labeling demonstrating CXCR4 labeled with FITC (green), B (i) and CD31 labeled with Texas red (red), B (ii). Colocalization of CXCR4 and CD31 was viewed as yellow under confocal microscopy, B (iii). C, Skeletal muscle homogenates from both control and CLI groups were analyzed by Western blot analysis. The samples were run individually for each of the patient samples, and a representative figure shown. Compared with controls (n = 12), the CLI group (n = 12) showed increased CXCL12α (control vs CLI; 3.14 ± 0.34 vs 8.77 ± 1.48 RDU, P < .01), C (i) and CXCR4 (control vs CLI; 1.50 ± 0.31 vs 5.41 ± 0.87 RDU, P < .01), C (iii) expressions. There was no significant difference in CXCL12β expression between the two groups, C (ii), (control vs CLI; 0.96 ± 0.14 vs 1.09 ± 0.16 RDU, P = .06). β−actin was used as a control for sample loading, C (iv). Values are expressed as mean ± SD. Error bars represent standard deviation. RDU, relative density unit. Scale bar = 100 μm.