Biochemically engineered stromal cell-derived factor 1-alpha analog increases perfusion in the ischemic hind limb

Abstract

Background: Despite promising therapeutic innovation over the last decade, peripheral arterial disease remains a prevalent morbidity, as many patients are still challenged with peripheral ischemia. We hypothesized that delivery of engineered stromal cell-derived factor 1-alpha (ESA) in an ischemic hind limb will yield significant improvement in perfusion.

Methods: Male rats underwent right femoral artery ligation, and animals were randomized to receive a 100 μL injection of saline (n = 9) or 6 μg/kg dosage of equal volume of ESA (n = 12) into the ipsilateral quadriceps muscle. Both groups of animals were also given an intraperitoneal injection of 40 μg/kg of granulocyte macrophage colony-stimulating factor (GMCSF). Perfusion was quantified using a laser Doppler imaging device preoperatively, and on postoperative days 0, 7, and 14. Immunohistochemistry was performed to quantify angiogenesis on day 14, and an mRNA profile was evaluated for angiogenic and inflammatory markers.

Results: Compared with the saline/GMCSF group at day 14, the ESA/GMCSF-injected animals had greater reperfusion ratios (Saline/GMCSF, 0.600 ± 0.140 vs ESA/GMCSF, 0.900 ± 0.181; group effect P = .006; time effect P < .0001; group×time effect P < .0001), elevated capillary density (10×; Saline/GMCSF, 6.40 ± 2.01 vs ESA/GMCSF, 18.55 ± 5.30; P < .01), and increased mRNA levels of vascular endothelial growth factor-A (Saline/GMCSF [n = 6], 0.298 ± 0.205 vs ESA/GMCSF [n = 8], 0.456 ± 0.139; P = .03).

Conclusions: Delivery of ESA significantly improves perfusion in a rat model of peripheral arterial disease via improved neovasculogenesis, a finding which may prove beneficial in the treatment strategy for this debilitating disease.

Fig 1

Crystallographic structural representation of SDF-1α and ESA. The N terminal (green), central region (yellow), and C terminal (magenta) are denoted by their respective colors. The central β- sheet region (yellow) in SDF-1α is replaced by a diproline linker in ESA. The corresponding amino acid sequence of each molecule is also depicted and colored according to region. (From Hiesinger W, Perez-Aguilar JM, Atluri P, et al. Computational protein design to reengineer stromal cell-derived factor-1alpha generates an effective and translatable angiogenic polypeptide analog. Circulation 2011; 124:S18–26.)

Fig 2

Capillary density stratified by treatment group. A, Ligated hind limb samples were cryosectioned and stained for von Willebrand Factor (vWF), smooth muscle actin (SMA), and DAPI. The ESA/GMCSF group exhibited a significantly higher mean capillary density (n = 12, 18.55 ± 5.30) than that of the saline/GMCSF group (n = 9, 6.40 ± 2.01, *P < .01). Error bars denote SE. B, Representative fluorescent microscopy images of quadriceps sections at 10x magnification. Bar = 100μm.

Fig 3

Graph showing VEGFA-mRNA fluorescence ratio after normalizing to GAPDH. Flourescence was measured in both quadricep and calf samples. ESA/GMCSF-treated group showed significantly higher fluorescence ratios in both the quad and calf (n = 8, Quad: 0.456 ± 0.139, Calf: 0.473 ± 0.106) relative to the saline/GMCSF group (n = 6, Quad: 0.298 ± 0.205, Calf: 0.285 ± 0.136, *P = .03, **P = .04). Error bars denote SE.

Fig 4

mRNA levels of pro-inflammatory markers in quadriceps of ischemic limbs in both treatment groups. There was minimal difference between ESA/GMCSF (n = 8, IL-12B: 0.0021 ± 0.00067, IL-2: 0.0014 ± 0.00021, IL-1Beta: 0.0018 ± 0.00042, IL-10: 0.0020 ± 0.00064, IFN-gamma: 0.0018 ± 0.00031, IL-1alpha: 0.0014 ± 0.00036, IL-6: 0.0024 ± 0.00061, IL-4: 0.00084 ± 0.00014) versus saline/GMCSF groups (n = 6, IL-12B: 0.0020 ± 0.00058, P = .41, IL-2: 0.0014 ± 0.00050, P = .39, IL-1Beta: 0.0021 ± 0.00065, P = .21, IL-10: 0.0020 ± 0.00081, P = .40, IFN-gamma: 0.0015 ± 0.00062, P = .16, IL-1alpha: 0.0020 ± 0.0010, P = .074, IL-6: 0.0019 ± 0.00047, P = .085, IL-4: 0.0011 ± 0.00038, P = .025). Error bars denote SE.

Fig 5

A, Representative laser Doppler images of the preoperative hind limb and again at the study endpoint for each group. B, Graph depicting the ratio of perfusion in the ischemic hind limb relative to the non-ligated hind limb pre-operatively and at three time points after induced ischemia. The ESA/GMCSF group (n = 12, 0.900 ± 0.181) showed marked perfusion augmentation by Day 14 relative to that of the saline/GMCSF group (n = 9, 0.600 ± 0.140, group effect: P = .006, time effect: P < .0001, group*time effect: P < .0001). Error bars denote SE.